G-EXJ-1030713 May 2012 DIAGNOSTICS SLIDE DECK. 2 G-EXJ-1030713 May 2012 TABLE OF CONTENTS ● Use of...

G-EXJ-1030713 May 2012 DIAGNOSTICS SLIDE DECK

2 G-EXJ-1030713 May 2012 TABLE OF CONTENTS Use of MRI in evaluating liver iron loading (and monitoring therapy) Cardiac MRI Cytogenetic Assessment in MDS Transcranial Doppler Ultrasonography (TCD) for assessment of stroke risk in Sickle Cell Disease Glossary of terms

G-EXJ-1030713 May 2012 USE OF MRI IN EVALUATING LIVER IRON LOADING (AND MONITORING THERAPY) NOTE: These slides are for use in educational oral presentations only. If any published figures/tables from these slides are to be used for another purpose (e.g. in printed materials), it is the individuals responsibility to apply for the relevant permission. Specific local use requires local approval

4 G-EXJ-1030713 May 2012 Outline Introduction to iron and liver iron overload Key methods for assessing liver iron liver biopsy SF SQUID liver MRI SIR method relaxometry methods (R2 and R2*) Clinical recommendations for measuring LIC Summary LIC = liver iron concentration; MRI = magnetic resonance imaging; SF = serum ferritin; SIR = signal intensity ratio; SQUID = superconducting quantum interface device.

G-EXJ-1030713 May 2012 Introduction to iron and iron overload

6 G-EXJ-1030713 May 2012 Iron overload Iron overload is common in patients who require intermittent or regular blood transfusions to treat anaemia and associated conditions it may be exacerbated in some conditions by excess gastrointestinal absorption of iron Iron overload can lead to considerable morbidity and mortality 1 Excess iron is deposited in major organs, resulting in organ damage the organs that are at risk of damage due to iron overload include the liver, heart, pancreas, thyroid, pituitary gland, and other endocrine organs 2,3 1 Ladis V, et al. Ann NY Acad Sci. 2005;1054:445-50. 2 Gabutti V, Piga A. Acta Haematol. 1996;95:26-36. 3 Olivieri NF. N Engl J Med. 1999;341:99-100.

7 G-EXJ-1030713 May 2012 Importance of analysing liver iron A patients LIC is the best measure of total body iron stores Knowing the liver iron concentration helps to predict the risk of hepatic and extra-hepatic complications 14 1 Batts KP. Mod Pathol. 2007;20:S31-9. 2 Jensen PD, et al. Blood. 2003;101:91-6. 3 Angelucci E, et al. Blood. 2002;100:17-21. 4 Telfer PT, et al. Br J Haematol. 2000;110:971-7.

8 G-EXJ-1030713 May 2012 LIC threshold of 7 mg Fe/g dry wt 0 5 10 15 20 25 All (n = 1,744) TM (n = 937) TI (n = 84) SCD (n = 80) Mean LIC + SD over previous year prior to enrolment in EPIC trial (mg Fe/g dry wt) Cappellini MD, et al. Blood. 2008;112:[abstract 3880]. Importance of analysing liver iron (cont.) All transfusion-dependent patients prior to study enrolment had moderate-to-severe hepatic iron loading

9 G-EXJ-1030713 May 2012 Overview of LIC correlations with other measurements DFS = disease-free survival. 1 Angelucci E, et al. N Engl J Med. 2000;343:327-31. 2 Jensen PD, et al. Blood. 2003;101:91-6. 3 Angelucci E, et al. Blood. 2002;100:17-21. 4 Telfer PT, et al. Br J Haematol. 2000;110:971-7. 5 Noetzli LJ, et al. Blood. 2008;112:2973-8. LIC Hepatocellular injury 2 and fibrosis 3 Body iron stores 1 Cardiac iron 5 Cardiac DFS 4

10 G-EXJ-1030713 May 2012 LIC prediction of total body iron stores BMT = bone marrow transplantation. 1 Olynyk JK, et al. Am J Gastroenterol. 1998;93:346-50. 2 Angelucci E, et al. N Engl J Med. 2000;343:327-31. Sample > 1 mg dry wt (n = 25) r = 0.98 0510152025 300 250 200 150 100 50 0 Body iron stores (mg/kg) LIC (mg Fe/g dry wt) Hereditary haemochromatosis 1 Iron removed (g) LIC (g/g) 0510152025 50,000 40,000 30,000 20,000 10,000 0 -TM 2 LIC is a reliable measure of total body iron stores in hereditary haemochromatosis and -TM

11 G-EXJ-1030713 May 2012 Serum ferritin measurement alone underestimates the body iron load Origa R, et al. Haematologica. 2007;92:583-8. Taher A, et al. Haematologica. 2008;93:1584-6. -TI -TM 05101520253035 LIC (mg Fe/g dry wt) SF ( g/L) 2,000 4,000 6,000 8,000 10,000 12,000 14,000 0 SF ( g/L) 05101520253035404550 LIC (mg Fe/g dry wt) 1,000 2,000 3,000 4,000 5,000 6,000 7,000 8,000 9,000 10,000 0 -TI -TM SF has almost no sensitivity or specificity for iron stores in thalassaemia intermedia

G-EXJ-1030713 May 2012 Assessing liver iron overload

13 G-EXJ-1030713 May 2012 Key methods for assessing liver iron Liver biopsy LIC advantages and disadvantages correlation of LIC with other measurements SF concentration over time advantages and disadvantages correlation of SF levels with other measurements SQUID advantages and disadvantages Liver MRI advantages and disadvantages relaxometry methods (T2 and T2*) SIR method Olivieri NF, Brittenham GM. Blood. 1997;89:739-61. Direct method Indirect methods

G-EXJ-1030713 May 2012 Liver biopsy

15 G-EXJ-1030713 May 2012 Technique for taking a percutaneous liver biopsy Step 1. The patient lies on his back, or his left side Step 4. The patient must hold breath for 5-10 seconds when the needle is quickly pushed in and out. As the needle comes out it brings with it a small sample of liver tissue Patient preparation: Blood tests are done shortly before the biopsy to check blood clotting time, to exclude risk of bleeding following the biopsy. The biopsy is commonly preceded by an ultrasound examination of the liver to determine the best and safest biopsy site Step 3. A special hollow needle is inserted into the liver, usually between the 2 lower ribs on the right hand side Step 2. The place for the biopsy is cleaned with antiseptic and local anaesthesia is provided (s.c. on the right hand side) Liver biopsy A tiny incision is made between the ribs, and a needle is inserted to reach the area of the liver where a tissue sample is taken. The procedure requires local anaesthesia Area where a tissue sample is taken from Overall: The procedure is carried out by a qualified physician or surgeon in an outpatient care centre or hospital. It is fast (not longer than 5 min) and the patient is discharged shortly after adam.com

16 G-EXJ-1030713 May 2012 Processing the liver biopsy sample Gross histopathological examination reveals presence of abnormal cells or liver tissue used to determine presence and degree of cirrhosis and fibrosis LIC measurement by iron staining by atomic absorption spectroscopy: the current gold standard! Who does the test? preparation of the samples might be by a trained technician the analysis requires a qualified pathologist Angelucci E, et al. Haematologica. 2008;93:741-52. Image from: www.pathguy.com/lectures/cirrhosis_trichrome.jpg

17 G-EXJ-1030713 May 2012 Liver biopsy Liver biopsy with iron measurement by atomic absorption spectroscopy is the gold standard for measuring LIC 1 LIC threshold (mg Fe/g dry wt) 2 LIC threshold ( mol Fe/g dry wt) Clinical relevance 1.832Upper 95% of normal 15.0 269 Greatly increased risk of cardiac disease and early death 1 Angelucci E, et al. Haematologica. 2008;93:741-52. 2 St Pierre TG, et al. Blood. 2005;105:855-61.

18 G-EXJ-1030713 May 2012 Liver biopsy: pros and cons Pros 1 Cons Direct measurement of LIC Validated reference standard Quantitative, specific, and sensitive Allows for measurement of non-haem storage iron Provides information on liver histology/pathology Correlates with morbidity and mortality Invasive and painful procedure with risk of potentially serious complications 1 May involve sampling errors, especially in patients with cirrhosis 1 Requires skilled physicians 1 Laboratory techniques not standardized 1 iron measurement by atomic absorption spectroscopy 2 or chemical determination 3 wet or dry weight quoted iron concentration varies throughout the liver, 4 sample size often insufficient (requires 1 mg dry weight, or > 4 mg wet weight) 1 TIF. Guidelines for the Clinical Management of Thalassemia. 2nd rev. ed. Cyprus: TIF; 2008. Available from: www.thalassaemia.org.cy/pdf/Guidelines_2nd_revised_edition_EN.pdf. Accessed December 2010. 2 Angelucci E, et al. Haematologica. 2008;93:741-52. 3 Wood JC. Blood Rev. 2008;22 Suppl 2:S14-21. 4 Ambu R, et al. J Hepatol. 1995;23:544-9.

19 G-EXJ-1030713 May 2012 Heterogeneity of iron concentration throughout the liver From autopsy of a patient with beta-zero-thalassaemia. Ambu R, et al. J Hepatol. 1995;23:544-9. 020% 2040% 4060% 6080% 80100% Iron is unevenly distributed in the liver; therefore, a small sample may not give an absolutely representative mean LIC

G-EXJ-1030713 May 2012 SF Concentration

21 G-EXJ-1030713 May 2012 Ferritin and SF Ferritin is primarily an intracellular protein that stores iron in a form readily accessible to cells releases iron in a controlled fashion The molecule is shaped like a hollow sphere and it stores ferric (Fe 3+ ) iron in its central cavity the storage capacity of ferritin is approximately 4,500 Fe 3+ ions per molecule Ferritin is found in all tissues, though primarily in the liver, spleen, and bone marrow A small amount is also found in the blood as serum ferritin Harrison PM, Arosio P. Biochim Biophys Acta. 1996;1275:161-203. SF > 1,000 g/L is a marker of excess body iron

22 G-EXJ-1030713 May 2012 SF: pros and cons SF levels from a blood sample are measured ProsCons Easy to assess Inexpensive Positive correlation with morbidity and mortality Allows longitudinal follow-up of patients Indirect measurement of iron burden Fluctuates in response to inflammation, abnormal liver function, ascorbate deficiencies TIF. Guidelines for the Clinical Management of Thalassaemia. 2nd rev. ed. Cyprus: TIF; 2008. Available from: www.thalassaemia.org.cy/pdf/Guidelines_2nd_revised_edition_EN.pdf. Accessed December 2010.

G-EXJ-1030713 May 2012 SQUID

24 G-EXJ-1030713 May 2012 SQUID: superconducting quantum interference device Carneiro AA, et al. Reson Med. 2005;43:122-8. Magnetizing coil Dewar Liquid helium SQUID Pick to coil Water bag Patient Mattress Bed Piston H2OH2O Patient preparation: No special patient preparation is required. Ultrasound is used to evaluate the depth and size of the liver. The patient lies on their back with their torso surrounded by a 5-L water bag to minimize contributions from other tissues Step 2. LIC corresponds to the variation of magnetization detected and is calculated using custom-made Matlab 6.5 software Principle of the technique: Normal tissue is diamagnetic and has a magnetic susceptibility similar to that of water. In the presence of iron, tissue susceptibility is changed proportional to the amount of iron present. This alteration is detected, allowing non-invasive measurement of LIC Step 1. The susceptometer applies a low-power (114 T and 7.7 Hz) homogeneous magnetizing field in the hepatic region. Sensitive detectors measure the interference of tissue iron vs the water reference medium within the field Overall: The procedure is carried out by a qualified radiologist in a hospital. It is fast (not longer than 5 min) and the patient is discharged immediately after. Processing could be done on the spot and is faster then LIC histopathological examination

25 G-EXJ-1030713 May 2012 SQUID: pros and cons ProsCons Non-invasive 1 Wide linear range 1 Good correlation with LIC by biopsy 2 Requires expensive, specialized equipment and expertise 1 Not widely available 1 Each machine should be individually calibrated 1 SQUID can underestimate LIC 3 1 TIF. Guidelines for the Clinical Management of Thalassaemia. 2nd rev. ed. Cyprus: TIF; 2008. Available from: www.thalassaemia.org.cy/pdf/Guidelines_2nd_revised_edition_EN.pdf. Accessed December 2010. 2 Sheth S. Pediatr Radiol. 2003;33:373-7. 3 Piga A, et al. Blood. 2005;106:[abstract 2689]. 250 200 150 100 50 0 0 100150200250 Hepatic iron (magnetic) ( mol Fe/g wet wt) Hepatic iron (biopsy) ( mol Fe/g wet wt) R = 0.99 p < 0.001 SQUID is a non-invasive method that has been calibrated, validated, and used in clinical studies, but the complexity, cost and technical demands limit its use

G-EXJ-1030713 May 2012 Liver MRI

27 G-EXJ-1030713 May 2012 MRI ROI = region of interest; SI = signal intensity; TE = echo time. Brittenham GM, Badman DG. Blood. 2003;101:15-9. Ridgway JP. J Cardiovasc Magn Reson. 2010;12:71. Integral radiofrequency transmitter (body) coil Main magnet coils x,y,z gradient coils Patient table Patient preparation: All infusion and medication pumps should be removed. The scan does not require contrast agent, and so no peripheral vein access is needed Step 2. Post-processing: As TE increases, the images SI decreases. The relationship between TE and SI in a selected part of the image (i.e. ROI) is analysed with specialized software or manually. Data are reported as relaxation times (T2 or T2*), depending on the acquisition method Principle of the technique: A strong magnetic field is used to organize the protons in the tissue in 1 direction. Then radiofrequency is used to knock them off. The time for them to re-align with the magnetic field and the energy they release during the process depend on the interactions of the proton with other ions, notably iron ions. These events could be measured at various TEs and then analysed to reveal the iron content in the tissue Step 1. Image acquisition: Images are taken at various TEs Overall: The procedure is carried out by a qualified radiologist in a hospital. Acquisition is fast (approx. 5 min), and the patient is discharged immediately after. Processing may require specialized software and is done afterwards

28 G-EXJ-1030713 May 2012 MRI is increasingly being used as a non-invasive method to measure LIC ProsCons Non-invasive 1,2 Assesses iron content throughout the liver 2 Increasingly and widely available worldwide 2 Pathological status of liver and heart can be assessed in parallel 2 Validated relationship with biopsy LIC 3 6 Indirect measurement of LIC 2 Requires MRI with dedicated imaging method 2 Sensitivity depends on type of scanner, degree of iron overload, presence of fibrosis, and inflammation 7 1 Chavhan GB, et al. Radiographics. 2009;29:1433-49. 2 TIF. Guidelines for the Clinical Management of Thalassaemia. 2nd rev. ed. Cyprus: TIF; 2008. Available from: www.thalassaemia.org.cy/pdf/Guidelines_2nd_ revised_edition_EN.pdf. Accessed December 2010. 3 Christoforidis A, et al. Eur J Haematol. 2009;82:388-92. 4 St Pierre TG, et al. Blood. 2005;105:855-61. 5 Wood JC, et al. Blood. 2005;106:1460-5. 6 Hankins JS, et al. Blood. 2009;113:4853-5. 7 Sirlin CB, Reeder SB. Magn Reson Imaging Clin N Am. 2010;18:359-81.

29 G-EXJ-1030713 May 2012 MRI scanners Manufacturers Siemens Healthcare (Erlangen, Germany; www.siemensmedical.com) GE Healthcare (Milwaukee, WI, USA; www.gemedicalsystems.com) Philips Healthcare (Best, the Netherlands; www.medical.philips.com) Magnetic field strength most imaging is done on 1.5 T machines 3 T machines give better signal:noise ratio 1 worse susceptibility artefacts 1 The upper detection limit is halved, therefore it is too low for many patients 1 lower T2 and T2* values than 1.5 T machines 2 Liver package (including standard sequences and analysis of the data) is included in the software provided together with the MRI machine specialized LIC analysis software can be bought separately 1 Wood JC, Ghugre N. Hemoglobin. 2008;32:85-96. 2 Storey P, et al. J Magn Reson Imaging. 2007;25:540-7.

30 G-EXJ-1030713 May 2012 Overview of MRI techniques used to measure LIC DATA ACQUISITION DATA ANALYSIS MAJOR PROS AND CONS A combination of gradient and spin echos Free website + Fast acquisition Simple data analysis Limited sensitivity Reproducibility Gradient echo (same technique as cardiac iron measurement) (1 min) Manually (free xls sheet) or with dedicated software (e.g CMR tool 3,000 GBP per year) + Fast acquisition Correlates well with LIC Susceptible to artefacts Training needs Spin echo (15min) Done centrally by Resonance Health (300 USD per scan) + Gold Standard Little training need Longer data acquisition time Cost of analysis Signal Intensity Ratio (SIR) method (Gandon/Ernst) Relaxometry method R2*(T2*) R2(T2) (Ferriscan ) Liver MRI Technique

31 G-EXJ-1030713 May 2012 MRI measurement of LIC: techniques There are 2 broad groups of techniques SIR methods (Gandon et al. methods) relaxometry methods (FerriScan and T2* (R2*) methods) ProsCons SIR method Fast data acquisition Relatively simple algorithms and data analysis Can be used in scanners with different magnetic strengths (0.5, 1.0, 1.5 T) Limited range of sensitivity (upper limit is 21 mg Fe/g dry wt [380 mol/L]) Assumptions on reference tissue Not reliable in cirrhosis Smaller reproducibility Relaxometry method Greater range of sensitivity Does not rely on reference tissue assumptions T2* (or R2*) is very quick (requires a single breath-hold) Has only been calibrated at 1.5 T Takes longer to acquire data, when done as T2 (or R2) Argyropoulou MI, Astrakas L. Pediatr Radiol. 2007;37:1191-200. Gandon Y, et al. Lancet. 2004;363:357-62. St Pierre TG, et al. Ann N Y Acad Sci. 2005;1054:379-85. Wood JC. Curr Opin Hematol. 2007;14:183-90. Wood JC, et al. Blood. 2005;106:1460-5.

32 G-EXJ-1030713 May 2012 SIR methods Most common protocol includes 4-gradient echo sequences with different TEs 1 spin-echo sequence 0 100 300 400 200 0100200400300 Study group Validation group Biopsy LIC ( mol Fe/g dry wt) MRI LIC ( mol Fe/g dry wt) Gandon Y, et al. Lancet. 2004;363:357-62. 1. Patient preparation (5 min) 2. Image acquisition (approx. 5-20 min) 3. Data analysis (depends on experience)

33 G-EXJ-1030713 May 2012 SIR methods (cont.) The ROI is selected in the liver and the reference tissue (muscle or fat), in each image The SI of the liver region is divided by that of the reference tissue A calculation algorithm to assist has been developed for 0.5, 1.0, and 1.5 T MRI machines 1 1 Gandon Y. Available from: http://www.radio.univ-rennes1.fr/Sources/EN/HemoResult.html. Accessed December 2010. 1. Patient preparation (5 min) 2. Image acquisition (approx. 5-20 min) 3. Data analysis (relatively fast)

34 G-EXJ-1030713 May 2012 Relaxometry methods: T2, T2*, T2, R2, and R2* If a spin-echo sequence is used, the relaxation time is T2 If a gradient-echo sequence is used, it is T2* These are related by the equation 1 1/T2* = 1/T2 + 1/T2 T2 is the magnetic field inhomogeneity of the tissue To attain a positive linear relationship with HIC T2* can be transformed into reciprocal R2*: R2* [Hz] = 1,000/T2* [ms] T2 can be transformed into reciprocal R2: R2 [Hz] = 1,000/T2 [ms] 1 Anderson LJ, et al. Eur Heart J. 2001;22:2171-9. 2 Wood JC, Ghugre N. Hemoglobin. 2008;32:85-96.

35 G-EXJ-1030713 May 2012 Relaxometry methods: R2 and R2* Several pulse sequences are included in the MRI software package Parameters R2 (for FerriScan ) spin echo sequence T2* (and R2*) gradient echo sequence FOV (mm)300 x 225350 x 300 Matrix (lines)256 x 176128 x 80 Resolution (mm)1.17 x 1.28 x 5.02.73 x 3.75 x 10.0 TR (ms)2500200 TE (ms)6, 9, 12, 15, 18Minimum possible (ideally < 2.0 ms) NEX (n)11 Flip angle ()9020 BW (Hz/px)3001,950 Segments (n)8 FatSatOn Wood JC, Ghugre N. Hemoglobin. 2008;32:85-96.

36 G-EXJ-1030713 May 2012 1 Wood JC, et al. Blood. 2005;106:1460-5. 2 St Pierre TG, et al. Blood. 2005;105:855-61. Biopsy LIC (mg Fe/g dry wt) Mean R2 (Hz) 300 250 200 150 100 50 0 Hepatitis Hereditary haemochromatosis -thalassaemia/Hb E -thalassaemia 010203040 Biopsy LIC (mg Fe/g dry wt) 350 300 250 200 150 100 50 0 0102030405060 R2 (Hz) LIC by biopsy, R = 0.98 Linear fit using biopsy data Controls, LIC by norms alone Correlation between R2-estimated LIC and LIC by biopsy R2-LIC calibration curve by St Pierre et al. 2005 2 R2-LIC calibration curve by Wood et al. 2005 1

37 G-EXJ-1030713 May 2012 Correlation between R2*-estimated LIC and LIC by biopsy 1 Wood JC, et al. Blood. 2005;106:1460-5. 2 Hankins JS, et al. Blood. 2009;113:4853-5. R2* (Hz) Biopsy LIC (mg Fe/g dry wt) R = 0.97 Patients Controls Fit 0 200 400 600 800 1,000 1,200 1,400 1,600 1,800 2,000 0102030405060 30 25 20 15 10 5 0 02004006008001000 R2*MRI (Hz) LIC (mg Fe/g dry wt) Correlation coefficient = 0.98 p < 0.001 R2*-LIC calibration curve by Hankins et al. 2 R2*-LIC calibration curve by Wood et al. 1

38 G-EXJ-1030713 May 2012 LIC estimated with R2 and R2* MRI correlate well with each other Wood JC, et al. Blood. 2005;106:1460-5. 0 10 20 30 40 50 01020304050 Estimated HIC (mg/dry) by R2-SP Estimated HIC (mg/dry) by R2* Patient data Linear fit, R=0.94

39 G-EXJ-1030713 May 2012 30 25 20 15 10 5 0 02004006008001000 Liver R2* (Hz) HIC (mg Fe/g of dry weight liver) Hankins, et al. Wood, et al. Anderson, et al. [Fe] (mg/g dry wt) Cardiac R2* (Hz) 0 2 4 6 8 10 12 14 0100200300400 R 2 = 0.82540 Liver MRICardiac MRI Gradient relaxometry (T2*, R2*) can conveniently measure cardiac and liver iron HIC = hepatic iron concentration Carpenter JP, et al. J Cardiovasc Magn Reson. 2009;11 Suppl 1:P224. Hankins et al Blood. 2009;113:4853-4855. Cardiac and liver iron can be assessed together conveniently by gradient echo during a single MRI measurement.

40 G-EXJ-1030713 May 2012 Relaxometry methods: pros and cons ProsCons R2* Correlate well to biopsy LIC 14 Greater sensitivity to iron deposits 5 Faster (images can be obtained in a single breath-hold) and easier 6 Can perform cardiac and liver iron assessment at the same time More susceptible to artefacts Requires expert training of a technician/ radiologist for data acquisition and data analysis R2 (Ferriscan ) Correlate well to biopsy LIC 14 Less affected by susceptibility artefacts 6 Highly sensitive and specific over a large range of LIC, including patients with severe haemosiderosis 7 The gold standard method in clinical trials Requires no training for data analysis (done centralized by Resonance Health) Multiple breath-holds required which increases MRI time Cost of analysis (300 USD per scan) 1 Christoforidis A, et al. Eur J Haematol. 2009;82:388-92. 2 St Pierre TG, et al. Blood. 2005;105:855-61. 3 Wood JC, et al. Blood. 2005;106:1460-5. 4 Hankins JS, et al. Blood. 2009;113:4853-5. 5 Anderson LJ, et al. Eur Heart J. 2001;22:2171-9. 6 Wood JC, Ghugre N. Hemoglobin. 2008;32:85-96. 7 Papakonstantinou, O, et al. J Magn Reson Imaging. 2009;29:853-9.

41 G-EXJ-1030713 May 2012 Relaxometry methods: R2 and R2* (cont.) Correct position is important so that the LIC across the whole liver can be measured Images are taken at various TEs Red line indicates correct position of the slice 1. Patient preparation (5 min) 2. Image acquisition (approx. 5-20 min) 3. Data analysis (depends on experience)

42 G-EXJ-1030713 May 2012 Liver R2* MRI Liver with normal iron levels Liver with severe iron overload Images courtesy of Dr J. de Lara Fernandes. T2* = 15.7 ms or R2* = 63.7 Hz or LIC = 1.3mg/g T2* = 1.1 ms or R2* = 909 Hz or LIC = 25.0 mg/g TE=1.3msTE=3.6ms TE=7.1ms TE=1.3msTE=3.6ms TE=7.1ms

43 G-EXJ-1030713 May 2012 How can I avoid artefacts when assessing LIC by MRI? When assessing LIC, one thing that is really important is to use fat saturation (usually automatically included in all the sequences). This is especially important if a patient has steatosis (e.g. adults with haemochromatosis) How frequent are artefacts in liver MRI? In contrast to cardiac MRI, the risk for motion artefacts (e.g. due to breathing) or susceptibility artefacts is much lower when performing liver MRI. As in cardiac MRI, if artefacts are present and too severe, scans may have to be repeated FAQ: artefacts Questions and answers were prepared under the review of Dr J. de Lara Fernandes, University of Campinas, Brazil.

44 G-EXJ-1030713 May 2012 Relaxometry methods: R2 and R2* (cont.) Determine ROI entire liver boundary, excluding obvious hilar vessels 1 Slice thickness varies, generally 515 mm 14 Number of slices anything from about 1 to 20 slices can be studied 14 Red outline shows position of ROI 1 Wood JC, et al. Blood. 2005;106:1460-5. 2 St Pierre TG, et al. Blood. 2005;105:855-61. 3 Papakonstantinou O, et al. J Magn Reson Imaging. 2009;29:853-9. 4 Hankins JS, et al. Blood. 2009;113:4853-5. 1. Patient preparation (5 min) 2. Image acquisition (approx. 5-20 min) 3. Data analysis (depends on experience)

45 G-EXJ-1030713 May 2012 Relaxometry methods: R2 and R2* (cont.) As TE increases, SI should decrease When plotted on a graph as iron load increases, the curve gets steeper T2 or T2* can be calculated from the curve R2 and R2* can also be calculated Calculations are done manually, or by specific licensed software (e.g. CMRtools ), or images could be directly sent to a validated centre performing FerriScan for analysis 100 80 60 40 20 0 1520 0 5 10 SI TE (ms) Typical non-iron-loaded tissue Increasing iron loading 1. Patient preparation (5 min) 2. Image acquisition (approx. 5-20 min) 3. Data analysis (depends on experience)

46 G-EXJ-1030713 May 2012 Analysis of the data The data can be analysed manually or using post-processing software ManuallyPost-processing software Excel spreadsheet ThalassaemiaTools (CMRtools) cmr 42 FerriScan MRmap MATLAB

47 G-EXJ-1030713 May 2012 Analysis of the data (cont.) MethodProsCons Excel spreadsheetLow cost Time-consuming Tedious ThalassaemiaTools (CMRtools) 1 Fast (1 min) 2 Easy to use FDA approved GBP 3,000 per year cmr 42(3) Easy to use FDA approved 3 Can generate T2*/R2* and T2/R2 maps with same software Allows different forms of analysis Generates pixel-wise fitting with colour maps 40,000 USD first year costs 12,000 USD per year after FDA = Food and Drug Administration. 1 www.cmrtools.com/cmrweb/ThalassaemiaToolsIntroduction.htm. Accessed Dec 2010. 2 Pennell DJ. JACC Cardiovasc Imaging. 2008;1:579-81. 3 www.circlecvi.com. Accessed Dec 2010.

48 G-EXJ-1030713 May 2012 MethodProsCons FerriScan 1 Centralized analysis of locally acquired data (206 active sites across 25 countries) Easy set-up on most MRI machines EU approved Validated on GE, Philips, and Siemens scanners USD 300 per scan Patients data are sent to reference centre MRmap 2 Uses IDL runtime, which is a commercial software (less expensive than cmr 42 /CMRtools) Can quantify T1 and T2 map with the same software Purely a research tool Not intended for diagnostic or clinical use MATLAB 3 Low costAvailable only locally Physicists or engineers need to write a MATLAB program for display and T2* measurement 1 www.resonancehealth.com/resonance/ferriscan. Accessed Dec 2010. 2 www.cmr-berlin.org/forschung/mrmapengl/index.html. Accessed Dec 2010. 3 Wood JC, Noetzli L. Ann N Y Acad Sci. 2010;1202:173-9. Analysis of the data (cont.)

49 G-EXJ-1030713 May 2012 What is truncation? After the selection of the ROI, the signal decay can be fitted using different models. In the truncation model, the late points in the curve (the plateau) are subjectively discarded to obtain a curve with an R 2 > 0.995. A new single exponential curve is made by fitting the remaining signals. What is the most frequent mistake made when interpreting the data from an MRI scan? Interpreting a liver MRI is more challenging than for a cardiac MRI, especially in patients with severe liver iron overload. Correcting the data using truncation analysis is very important (done automatically by some software). The example (see following slide) clearly shows what happens, if the truncation is not done correctly FAQ: mistakes in manual analysis of liver MRI data Questions and answers were prepared under the review of Dr J. de Lara Fernandes, University of Campinas, Brazil.

50 G-EXJ-1030713 May 2012 Non-truncated analysis with results with a poor R 2 (< 0.995). The apparent LIC of 4.65 suggests mild LICs. Observe the flat plateau of the data points after a TE of 3.62 ms The same patient, but analysing the data with only the 3 first data points results in a better (although not perfect) R 2. The LIC results in severe iron overload, reflecting the real concentrations of iron Analysis without truncation of the data Analysis with truncation of the data FAQ: mistakes in manual analysis of liver MRI data (cont.)

51 G-EXJ-1030713 May 2012 How to start measuring liver iron loading in a hospital? What steps need to be taken? To start assessing liver iron loading by MRI, these steps can be followed 1.Check MRI machine requirements 0.51.5 T (1.5 T is highly recommended for T2* and T2 calculations; 0.5 T only for SIR) calibrated includes a liver package 2.Optional: buy software for analysing the data (otherwise, Excel spreadsheet can be used) 3.Optional: training of personnel for acquiring MRI images 4.Optional: training of personnel on how to analyse the data FAQ: how to start measuring liver iron loading? Questions and answers were prepared under the review of Dr J. de Lara Fernandes, University of Campinas, Brazil.

52 G-EXJ-1030713 May 2012 LIC: interpretation of results LIC threshold values for classification of iron overload Iron levels LIC (mg Fe per g dry weight) LIC (mol Fe per g dry wt) R2 (s 1 ) R2* (s 1 )T2* (ms) Normal< 2< 35.6< 50< 88> 11.4 Mild overload 27 35.6 125.0 50 100 88 263> 3.8 11.4 Moderate overload 715 125 269 100 155 263 555> 1.8 3.8 Severe overload 15 269 155 555 1.8 Values estimated based on R2 LIC calibration curve; R2, R2* and T2* values valid for MRI machines with 1.5T only. St Pierre TG, et al. Blood 2005;105:855861; Wood JC, et al. Blood 2005;106:14601465.

53 G-EXJ-1030713 May 2012 Implementation of liver and cardiac MRI Slide presented at Global Iron Summit 2011 - With the permission of Juliano de Lara Fernandes 1.5T MRI Scanner Experienced radiologist Cardiac acquisition package Routine cardiac MR exams Post-processing analysis US$1.000.000 US$50.000 US$40.000 or US$4.000/y or in-house or outsource Yes No day training 1 day training Yes No 1-2 day training 4 day training Liver Analysis Liver Analysis Heart Analysis Heart Analysis

G-EXJ-1030713 May 2012 Summary

55 G-EXJ-1030713 May 2012 Summary Iron overload is a serious problem among patients who require blood transfusions to treat anaemia and associated conditions Analysing liver iron overload is important to predict risk of hepatic and extra-hepatic complications The extent of iron accumulation in the liver is a key prognostic indicator for morbidity and mortality MRI has the added advantage that iron levels throughout the liver can be analysed, rather than just the biopsied section (iron levels throughout the liver can vary) R2 is the most commonly used technique in clinical practice, although R2* is a comparable alternative across most ranges of iron overload and is faster

G-EXJ-1030713 May 2012 CARDIAC MRI Diagnostic Backgrounder NOTE: These slides are for use in educational oral presentations only. If any published figures/tables from these slides are to be used for another purpose (e.g. in printed materials), it is the individuals responsibility to apply for the relevant permission. Specific local use requires local approval

57 G-EXJ-1030713 May 2012 Outline Introduction to iron overload Assessing cardiac iron loading echocardiography cardiac MRI Cardiac MRI in practice preparation of the patient acquisition of the image analysis of the data Excel spreadsheet ThalassaemiaTools (CMRtools) cmr4 2 FerriScan MRmap MATLAB Summary MRI = magnetic resonance imaging.

G-EXJ-1030713 May 2012 Introduction to iron overload

59 G-EXJ-1030713 May 2012 Introduction to iron overload Iron overload is common in patients who require intermittent or regular blood transfusions to treat anaemia and associated conditions it may be exacerbated in some conditions by excess gastrointestinal absorption of iron Iron overload can lead to considerable morbidity and mortality 1 Excess iron is deposited in major organs, resulting in organ damage the organs that are at risk of damage due to iron overload include the liver, heart, pancreas, thyroid, pituitary gland, and other endocrine organs 2,3 1 Ladis V, et al. Ann NY Acad Sci. 2005;1054:445-50. 2 Gabutti V, Piga A. Acta Haematol. 1996;95:26-36. 3 Olivieri NF. N Engl J Med. 1999;341:99-109.

60 G-EXJ-1030713 May 2012 Importance of analysing cardiac iron In -thalassaemia major, cardiac failure and arrhythmia are risk factors for mortality 1 signs of myocardial damage due to iron overload: arrhythmia, cardiomegaly, heart failure, and pericarditis 2 heart failure has been a major cause of death in -thalassaemia patients in the past (5070%) 1,3 In MDS, the results of studies are less comprehensible the reported proportion of MDS patients with cardiac iron overload is inconsistent; from high to only a small proportion of MDS patients 47 cardiac iron overload occurs later than does liver iron overload 4,7,8 however, cardiac iron overload can have serious clinical consequences in MDS patients 1 Borgna-Pignatti C, et al. Haematologica. 2004;89:1187-93. 2 Gabutti V, Piga A. Acta Haematol. 1996;95:26-36. 3. Modell B, et al. Lancet. 2000;355:2051-2. 4 Jensen PD, et al. Blood. 2003;101:4632-9. 5 Chacko J, et al. Br J Haematol. 2007;138:587-93. 6 Konen E, et al. Am J Hematol. 2007;82:1013-6. 7 Di Tucci AA, et al. Haematologica. 2008;93:1385-8. 8 Buja LM, Roberts WC. Am J Med. 1971;51:209-21.

61 G-EXJ-1030713 May 2012 Baseline Latest follow-up p < 0.001 cT2* 20 ms cT2* < 10 ms Patients (%) cT2* = cardiac T2*. 1 Thomas AS, et al. Blood. 2010;116:[abstract 1011]. 2 Modell B, et al. Lancet. 2000;355:2051-2. Importance of analysing cardiac iron (cont.) In 2010, the overall mortality rate of -thalassaemia major patients in the UK was substantially lower than a decade ago (1.65 vs 4.3 per 1,000 patient years) 1,2 due to improved monitoring and management of iron overload over the last decade, 77% of patients have normal cardiac T2* 1 cardiac iron overload is no longer the leading cause of death in this population 1 60 17 23 7 0 10 20 30 40 50 60 70

62 G-EXJ-1030713 May 2012 Cardiac T2*: Overview of correlations with other measurements 1 Wood JC, et al. Blood. 2004;103:1934-6. 2 Anderson LJ, et al. Eur Heart J. 2001;22:2171-9. 3 Tanner MA, et al. J Cardiovasc Magn Reson. 2006;8:543-7. 4 Kirk P, et al. Circulation. 2009;120:1961-8. 5 Westwood MA, et al. J Magn Reson Imaging. 2005;22:229-33. For thalassaemia, but not sickle cell. APFR = atrial peak filling rate; EPFR = early peak filling rate; LIC = liver iron concentration; SF = serum ferritin. Weak or no correlation Transfusion duration 1 Ventricular dysfunction 1-3 Arrhythmia and heart failure 4 APFR EPFR:APFR 5 Need for cardiac medication 1-2 T2* SF and LIC 1-3

63 G-EXJ-1030713 May 2012 LVEF (%) 0 50 70 40 30 20 10 60 80 90 0 204060908010010305070 Cardiac T2* (ms) Cardiac T2* value of 37 ms in a normal heart Cardiac T2* value of 4 ms in a significantly iron-overloaded heart LVEF = left-ventricular ejection fraction. Anderson LJ, et al. Eur Heart J. 2001;22:2171-9. Normal T2* range Normal LVEF range Cardiac T2*: Relationship with LVEF Myocardial T2* values < 20 ms are associated with a progressive and significant decline in LVEF

64 G-EXJ-1030713 May 2012 0.1 Cardiac T2*: Relationship with cardiac failure and arrhythmia Kirk P, et al. Circulation. 2009;120:1961-8. T2* < 10 ms: relative risk 159 (p < 0.001) T2* < 6 ms: relative risk 268 (p < 0.001) Cardiac failure Proportion of patients developing cardiac failure Follow-up time (days) 600120180240300360 0.3 0.2 0 0.4 0.5 0.6 < 6 ms 68 ms 810 ms > 10 ms Arrhythmia 600120180240300360 0.15 0.10 0.05 0 0.20 0.25 0.30 < 10 ms 1020 ms > 20 ms T2* < 20 ms: relative risk 4.6 (p < 0.001) T2* < 6 ms: relative risk 8.65 (p < 0.001) Follow-up time (days) Proportion of patients with arrhythmia Low myocardial T2* predicts a high risk of developing cardiac failure and arrhythmia

G-EXJ-1030713 May 2012 Assessing cardiac iron overload

66 G-EXJ-1030713 May 2012 Assessing cardiac iron loading: Agenda Echocardiography Cardiac MRI advantages and disadvantages of cardiac MRI MRI: a non-invasive diagnostic tool T2* is the standard method for analysing cardiac iron

G-EXJ-1030713 May 2012 Echocardiography

68 G-EXJ-1030713 May 2012 Assessing cardiac iron loading: Echocardiography EF = ejection fraction. 1 Leonardi B, et al. JACC Cardiovasc Imaging. 2008;1:572-8. 2 Hoffbrand AV. Eur Heart J. 2001;22:2140-1. ProsCons Readily available 1 Relatively inexpensive 1 Does not detect early damage 2 Echocardiographic diastolic function parameters correlate poorly with LVEF and T2* 1 Cannot directly or indirectly quantify cardiac iron levels

G-EXJ-1030713 May 2012 Cardiac MRI

70 G-EXJ-1030713 May 2012 MRI: A non-invasive diagnostic tool Indirectly measures levels of iron in the heart MRI measures longitudinal (T1) and transverse (T2) relaxation times of the protons iron deposition disrupts the homogeneous magnetic field and shortens T1 and T2 times in a concentration-dependent manner RF = radio-frequency. 1 Wood JC, Ghugre N. Hemoglobin. 2008;32:85-96. 2 Wood JC, et al. Circulation. 2005;112:535-43. 3 Wang ZJ, et al. Radiology. 2005;234:749-55. 4 Ghugre NR, et al. Magn Reson Med. 2006;56:681-6. Protons Magnetic field RF/spin echo/gradient echo Echo signal T1, T2 Signal processing Iron

71 G-EXJ-1030713 May 2012 MRI: A non-invasive diagnostic tool (cont.) If a spin-echo sequence is used, the relaxation time is T2 If a gradient-echo sequence is used, it is T2* Cardiac MRI methods gradient-echo T2* MRI: most used in clinical practice spin-echo T2 MRI: less useful (motion artefacts common due to characteristics of the heart) TE = echo time. Adapted from Wood JC, Ghugre N. Hemoglobin. 2008;32:85-96. Protons Magnetic field Most used in clinical practice: Gradient echo Image acquired at different TEs Excel or software T2* [ms} R2* [Hz]= 1,000/T2* Spin echo Image acquired at different TEs Excel or software T2* [ms} R2* [Hz]= 1,000/T2*

72 G-EXJ-1030713 May 2012 Assessing cardiac iron loading: Cardiac MRI Advantages of MRIDisadvantages of MRI Non- invasive Rapidly assesses iron content in the septum of the heart Relative iron burden can be reproducibly estimated Functional parameters can be examined concurrently (e.g. LVEF) Iron status of liver and heart can be assessed in parallel Allows longitudinal follow-up Good correlation with morbidity and mortality outcomes Indirect measurement of cardiac iron Requires MRI imager with dedicated imaging method Relatively expensive and varied availability

73 G-EXJ-1030713 May 2012 What are sequences? Sequences are a set of radio-frequency and gradient pulses (slight tilts in the magnetization curves of the scanner) generated repeatedly during the scan, which produce echoes with varied amplitudes and shapes that will define the MR image What is gradient echo? A gradient-echo sequence is obtained after 2 gradient impulses are applied to the body, resulting in a signal echo that is read by the coils. In these sequences, the spins are not refocused and, therefore, are subject to local inhomogeneities, with a more rapid decay curve. For gradient-echo pulse sequences, the T2* relaxation times (which reflect these inhomogeneities) on the signal are more significant 1 Image from Ridgway JP. J Cardiovasc Magn Reson. 2010;12:71. FAQ: Cardiac MRI

74 G-EXJ-1030713 May 2012 Gradient relaxometry (T2*, R2*) is the method for analysing cardiac iron levels 1 Guo H, et al. J Magn Reson Imaging. 2009;30:394-400. 2 Anderson LJ, et al. Eur Heart J. 2001;22:2171-9. 3 Wood JC, Noetzli L. Ann N Y Acad Sci. 2010;1202:173-9. 4 Wood JC, Ghugre N. Hemoglobin. 2008;32:85-96. 5 Westwood M, et al. J Magn Reson Imaging. 2003;18:33-9. 6 Hoffbrand AV. Eur Heart J. 2001;22:2140-1. 7 He T, et al. Magn Reson Med. 2008;60:1082-9. T2* (gradient echo)T2 (spin echo) ProsGreater sensitivity to iron deposition 2 Shorter acquisition time 1 Less affected by motion artefacts 3 More readily available 3 Easier to perform 4 Good reproducibility 5 Less affected by susceptibility artefacts 1, due to metal implants, airtissue interfaces, proximity to cardiac veins ConsMore sensitive to static magnetic field inhomogeneity 1 Noise, motion, and blood artefacts can complicate analysis (particularly in heavily iron-loaded hearts) 7 Lack of sensitivity 6 Motion artefacts 6 Poor signal-to-background noise ratios at longer TEs 6 Longer acquisition time 1

75 G-EXJ-1030713 May 2012 HIC = hepatic iron concentration Carpenter JP, et al. J Cardiovasc Magn Reson. 2009;11 Suppl 1:P224. Hankins et al Blood. 2009;113:4853-4855. 30 25 20 15 10 5 0 02004006008001000 Liver R2* (Hz) HIC (mg Fe/g of dry weight liver) Hankins, et al. Wood, et al. Anderson, et al. [Fe] (mg/g dry wt) Cardiac R2* (Hz) 0 2 4 6 8 10 12 14 0100200300400 R 2 = 0.82540 Liver MRI Cardiac MRI Gradient relaxometry (T2*, R2*) can conveniently measure cardiac and liver iron Cardiac and liver iron can be assessed together conveniently by gradient echo during the a single MRI measurement.

76 G-EXJ-1030713 May 2012 Cardiac T2* MRI is usually measured in the septum of the heart Heart with normal iron levels Heart with severe iron overload Images courtesy of Dr J. de Lara Fernandes. T2* = 22.8 ms or R2* = 43.9 Hz T2* = 5.2 ms or R2* = 192 Hz

77 G-EXJ-1030713 May 2012 Conversion from T2* to R2* is a simple mathematical calculation: R2* = 1,000/T2* Level of cardiac iron overloadT2*, msR2*, Hz Normal 20 1 < 50 Mild, moderate1020 1 50100 Severe< 10 2 > 100 1 Anderson LJ, et al. Eur Heart J. 2001;22:2171-9. 2 Kirk P, et al. Circulation. 2009;120:1961-8. These values are only applicable to 1.5 T scanners 1 What is R2*?

78 G-EXJ-1030713 May 2012 Why should the data be presented as R2* and not T2*? Seven whole hearts from patients with transfusion-dependent anaemias were assessed by histology and cardiac MRI [Fe] (mg/g dry wt) Cardiac T2* (ms) [Fe] (mg/g dry wt) 0 2 4 6 8 10 12 14 010203040506070 R 2 = 0.949 Cardiac R2* (Hz) 0 2 4 6 8 10 12 14 0100200300400 R 2 = 0.82540 Carpenter JP, et al. J Cardiovasc Magn Reson. 2009;11 Suppl 1:P224. R2* has a linear relationship with tissue iron concentration, which simplifies the interpretation of data and allows comparison of changes over time

79 G-EXJ-1030713 May 2012 Anderson LJ, et al. Eur Heart J. 2001;22:2171-9. Hockey stick effect?Or a more gradual relationship? The relationship between cardiac T2*/R2* and LVEF Heart T2* (ms) LVEF (%) R2* (s 1 ) LVEF (%) 90 80 70 60 50 40 30 20 10 0 2030405060708090100 0 80 60 40 20 0 050100150200250 Why should the data be presented as R2* and not T2*? (cont.) R2* allows demonstration of cardiac risk in a more gradual way

80 G-EXJ-1030713 May 2012 Transform to R2* Standard errors on a single measurement are approximately constant with R2*, but are non-uniform with T2* Westwood M, et al. J Magn Reson Imaging. 2003;18:33-9. 60 50 40 30 20 10 0 0 2030405060 T2* second measurement (ms) T2* first measurement (ms) 120 100 80 60 40 20 0 0 406080100120 R2* second measurement (s 1 ) R2* first measurement (s 1 ) Why should the data be presented as R2* and not T2*? (cont.) R2* has a constant standard error that makes assessment of the significance of changes easier

G-EXJ-1030713 May 2012 Cardiac T2* MRI in practice

82 G-EXJ-1030713 May 2012 MRI scanners Manufacturers Siemens Healthcare (Erlangen, Germany; www.siemensmedical.com) GE Healthcare (Milwaukee, WI, USA; www.gemedicalsystems.com) Philips Healthcare (Best, the Netherlands; www.medical.philips.com) Magnetic field T2* varies with magnetic field strength 1 need 1.5 T for cutoff levels of 20 ms (iron overload) and 10 ms (severe iron overload) 1,2 Cardiac package needs to be acquired separately from the manufacturers. The cost is about USD 40,000. However, in most centres, this is available since MRI is frequently used in non-iron-related cardiovascular imaging includes all necessary for acquisition of the image sequences are included in Siemens and Philips Healthcare cardiac packages, but for GE Healthcare they need to be acquired separately (note: variations may exist between countries) 1 Anderson LJ, et al. Eur Heart J. 2001;22:2171-9. 2 Kirk P, et al. Circulation. 2009;120:1961-8.

83 G-EXJ-1030713 May 2012 Cardiac T2* MRI in practice: The process T2*, R2* *Time to manually calculate T2*/R2* values in an Excel spreadsheet depends on the experience of the physician. 1. Patient preparation (5 min) 2. Acquisition of the MRI image (approx. 5-20 min) 3. Analysis of MRI data (time depends on experience*)

84 G-EXJ-1030713 May 2012 Cardiac T2* MRI in practice: The process (cont.) Preparation of the patient Acquisition of the image Analysis of the data (post-processing) Excel spreadsheet ThalassaemiaTools, CMRtools cmr 42 FerriScan MRmap MATLAB

G-EXJ-1030713 May 2012 Preparation of the patient

86 G-EXJ-1030713 May 2012 Preparation of the patient Standard precautions need to be taken There is no need for peripheral vein access since no contrast agent is required Special care remove all infusion/medication pumps (e.g. with insulin, pain-relieving drugs) stop continuous i.v. application of ICT during the measurement ECG signal should be positioned according to scanner specifications ECG = electrocardiography.

G-EXJ-1030713 May 2012 Acquisition of the image

89 G-EXJ-1030713 May 2012 Acquisition of the image: MRI pulse sequences Pulse sequences are a preselected set of defined radio-frequency and gradient pulses are computer programs that control all hardware aspects of the scan determine the order, spacing, and type of radio-frequency pulses that produce magnetic resonance images according to changes in the gradients of the magnetic field Several different pulse sequences exist 1 a gradient-echo sequence generates T2* 1 Wood JC, Ghugre N. Hemoglobin. 2008;32:85-96.

90 G-EXJ-1030713 May 2012 1 Anderson LJ, et al. Eur Heart J. 2001;22:2171-9. 2 Westwood M, et al. J Magn Reson Imaging. 2003;18:33-9. 3 He T, et al. J Magn Reson Imaging. 2007;25:1205-9. 4 He T, et al. Magn Reson Med. 2008;60:1082-9. 5 Pepe A, et al. J Magn Reson Imaging. 2006;23:662-8. SequenceGroup Number of echoes per breath-hold Heart regions Pre- pulse RR intervals TR Bright blood (Anderson et al.) 1 London (Pennell) 1 (but multiple breath-holds) 1 (septum)No1Variable Novel bright blood (Westwood et al) 2 London (Pennell) Multiple1 (septum)No1Fixed Black blood (He et al) 3-4 London (Pennell) Multiple1 (septum)Yes2Fixed Multi-slice (Pepe et al) 5 Pisa (Pepe) Multiple Multi- region No1Fixed The most common commercially available T2* acquisition techniques The various techniques give clinically comparable results. 2-3, 5

91 G-EXJ-1030713 May 2012 Acquisition of the image: TEs Images are taken at a minimum of 5 different TEs, normally 8 12 1 The choice of minimum TE determines the smallest measurable T2 1 ideally, min TE 2 ms, max TE 17 20 ms Different T2* acquisition techniques according to TE multiple breath-hold: acquire an image for each TE in separate breath-holds 2 single breath-hold multi-echo acquisition: acquire images for all TE during 1 breath-hold 3 Mean R2* compared with true value in the case of synthetic images for different minimum TEs, but same echo duration (18 ms) 4 1 Wood JC, Noetzli L. Ann N Y Acad Sci. 2010;1202:173-9. 2 Anderson LJ, et al. Eur Heart J. 2001;22:2171-9. 3 Westwood M, et al. J Magn Reson Imaging. 2003;18:33-9. 4 Ghugre NR, et al. J Magn Reson Imaging. 2006;23:9-16. 500 450 400 350 300 250 200 150 100 50 0 0100200300400500 True R2* (Hz) Mean R2*: ramp, dualtone, & uniform (Hz) Shortest TE = 2 ms Shortest TE = 1 ms Shortest TE = 4 ms Shortest TE = 5.5 ms True

92 G-EXJ-1030713 May 2012 How does the MRI data output looks like? Data visualizationMRI data output 1 Wood JC, Ghugre N. Hemoglobin. 2008;32:85-96. During a single breath hold the pulse sequence run several times at increasing echo time (TE), generating data points corresponding to decreased signal intensity 1 FrameTE (ms)Mean ST 01.989.5 13.683.6 25.376.8 37.070.6 48.764.5 510.459.2 612.254.9 713.950.2 815.645.8 917.342.4

93 G-EXJ-1030713 May 2012 Which is recommended: single or multiple breath-hold technique? Comparison of the 2 methods, single and multiple breath-hold, showed no significant skewing between T2* values in all patients with -thalassaemia major, regardless of their T2* value (see Bland- Altman plots) 1 However, in cardiac MRI the most recommended technique is single breath-hold, because it allows quick acquisition of the information. This is especially important to avoid movement artefacts (heart beating, breathing) and assure the good quality of the MRI image 1 Westwood M, et al. J Magn Reson Imaging. 2003;18:33-9. Patients with T2* < 20 ms 1 Patients with T2* 20 ms 1 FAQ: Acquisition technique

94 G-EXJ-1030713 May 2012 Acquisition of the image Single breath-hold multi-echo acquisition take a short-axis slice of the ventricle (halfway between the base and the apex): orange line image acquisition should occur immediately after the R wave do not alter any settings that could alter TE (e.g. FOV) Image courtesy of Dr J. de Lara Fernandes.

G-EXJ-1030713 May 2012 Analysis of the data (post-processing)

97 G-EXJ-1030713 May 2012 How T2* is calculated from the MRI output? Data visualization 1 Wood JC, Ghugre N. Hemoglobin. 2008;32:85-96. Curve Fitting T2* Noise level T2* calculation is fitting a curve on the data points and calculating at what echo time no signal is left from iron (only noise) 1

98 G-EXJ-1030713 May 2012 Analysis of the data The data can be analysed manually or using post-processing software ManuallyPost-processing software Excel spreadsheet ThalassaemiaTools (CMRtools) cmr 42 FerriScan MRmap MATLAB

99 G-EXJ-1030713 May 2012 Analysis of the data (cont.) MethodProsCons Excel spreadsheetLow costTime-consuming Tedious ThalassaemiaTools (CMRtools) 1 Fast (1 min) 2 Easy to use FDA approved GBP 3,000 per year cmr 42(3) Easy to use FDA approved 3 Can generate T2*/R2* and T2/R2 maps with same software Allows different forms of analysis Generates pixel-wise fitting with colour maps 40,000 USD first year costs 12,000 USD per year after FDA = Food and Drug Administration. 1 www.cmrtools.com/cmrweb/ThalassaemiaToolsIntroduction.htm. Accessed Dec 2010. 2 Pennell DJ. JACC Cardiovasc Imaging. 2008;1:579-81. 3 www.circlecvi.com. Accessed Dec 2010.

100 G-EXJ-1030713 May 2012 MethodProsCons FerriScan 1 Centralized analysis of locally acquired data (206 active sites across 25 countries) Easy set-up on most MRI machines EU approved Validated on GE, Philips, and Siemens scanners USD 100 per scan Patients data are sent to reference centre MRmap 2 Uses IDL runtime, which is a commercial software (less expensive than cmr 42 /CMRtools) Can quantify T1 and T2 map with the same software Purely a research tool Not intended for diagnostic or clinical use MATLAB 3 Low costAvailable only locally Physicists or engineers need to write a MATLAB program for display and T2* measurement 1 www.resonancehealth.com/resonance/ferriscan. Accessed Dec 2010. 2 www.cmr-berlin.org/forschung/ mrmapengl/index.html. Accessed Dec 2010. 3 Wood JC, Noetzli L. Ann N Y Acad Sci. 2010;1202:173-9. Analysis of the data (cont.)

101 G-EXJ-1030713 May 2012 What are the most common mistakes in analysing the data that could lead to a wrong interpretation of the T2* value? Interpreting the data from cardiac MRI is usually quite straightforward; problems may arise when analysing data from patients with severe cardiac iron overload. In this case, the signal from heavily iron-loaded muscle will decay quickly and a single exponential decay curve does not fit the data well. 1 Models exist that can help to solve this issue (see next slide): 1. the offset model (Prof Wood and colleagues) 2. truncation of the data (Prof Pennell and colleagues) Both models should give comparable results; the differences should not be clinically relevant 1 Wood JC, Noetzli L. Ann N Y Acad Sci. 2010;1202:173-9. 2 Ghugre NR, et al. J Magn Reson Imaging. 2006;23:9-16. Signal decay curve from a patient with T2* 5 ms, showing that the data do not fit well 2 FAQ: Mistakes in analysing the data

102 G-EXJ-1030713 May 2012 What is truncation? After the selection of the ROI, the signal decay can be fitted using different models. In the truncation model, the late points in the curve that form a plateau are subjectively discarded; the objective is to have a curve with an R 2 > 0.995. A new single exponential curve is made by fitting the remaining signals. 1 Generally, a truncation model should be used with the bright-blood technique to obtain more reproducible and more accurate T2* measurements 1 What is an offset model? The offset model consists of a single exponential with a constant offset. Using only the exponential model can underestimate the real T2* values (at quick signal loss at short TE, there is a plateau), while inclusion of the offset model into the fitting equation can improve this. 2 Generally, the offset model is recommended to be used with the black-blood technique 1 He T, et al. Magn Reson Med. 2008;60:1082-9. 2 Ghugre NR, et al. J Magn Reson Imaging. 2006;23:9-16. FAQ: Mistakes in analysing the data (cont.)

103 G-EXJ-1030713 May 2012 How to start measuring cardiac iron loading in a hospital? What steps need to be taken? To start assessing cardiac iron loading by MRI, these steps can be followed: 1.Check MRI machine requirements 1.5 T calibrated 2.Buy cardiac package from the manufacturer. It must include all that is necessary for acquisition of the data (the sequences are included with Siemens and Philips Healthcare cardiac packages, but for GE Healthcare they need to be acquired separately) 3.Optional: buy software for analysing the data (if not, Excel spreadsheet can be used) 4.Highly recommended: training of personnel for acquisition of cardiac MR images (e.g. functional analyses) 5.Highly recommended: training of personnel on how to analyse the data with the chosen software FAQ: How to start measuring cardiac iron loading?

104 G-EXJ-1030713 May 2012 Implementation of liver and cardiac MRI 1.5T MRI Scanner Experienced radiologist Cardiac acquisition package Routine cardiac MR exams Post-processing analysis US$1.000.000 US$50.000 US$40.000 or US$4.000/y or in-house or outsource Yes No day training 1 day training Yes No 1-2 day training 4 day training Liver Analysis Liver Analysis Heart Analysis Heart Analysis Slide presented at Global Iron Summit 2011 - With the permission of Juliano de Lara Fernandes

106 G-EXJ-1030713 May 2012 Summary Iron overload is common in patients who require intermittent or regular blood transfusions to treat anaemia and associated conditions Analysing cardiac iron levels is important in -thalassaemia major, cardiac failure and arrhythmia are risk factors for mortality in MDS, cardiac iron overload can have serious clinical consequences due to improved monitoring and management of iron overload over the last decade, 77% of patients have normal cardiac T2* 1 MRI: the method to rapidly and effectively assess cardiac iron loading T2* allows specific assessment of cardiac iron levels. The use of this convenient, non-invasive procedure has had a significant impact on outcomes in patients with cardiac iron overload 1 R2* is a simple calculation from T2* and has a linear relationship with cardiac iron concentration 1 Thomas AS, et al. Blood. 2010;116:[abstract 1011]. 2 Modell B, et al. J Cardiovasc Magn Reson. 2008;10:42-9.

G-EXJ-1030713 May 2012 CYTOGENERIC ASSESSMENT IN MDS NOTE: These slides are for use in educational oral presentations only. If any published figures/tables from these slides are to be used for another purpose (e.g. in printed materials), it is the individuals responsibility to apply for the relevant permission. Specific local use requires local approval.

108 G-EXJ-1030713 May 2012 Outline MDS Classification and prognosis scoring Practical guide to bone marrow aspirate analysis in MDS Cytogenetics of MDS in bone marrow aspirates Summary LIC = liver iron concentration; MRI = magnetic resonance imaging; SF = serum ferritin; SIR = signal intensity ratio; SQUID = superconducting quantum interface device.

109 G-EXJ-1030713 May 2012 What are the myelodysplastic syndromes (MDS)? MDS are a spectrum of heterogeneous myeloid clonal disorders Occurrence: De novo (primary MDS) Secondary or treatment-related MDS MDS are associated with significant morbidity and mortality due to: Risk of transformation to acute myeloid leukemia (AML) Cytopenias Impaired quality of life (frequent transfusions, iron overload, etc)

110 G-EXJ-1030713 May 2012 Common features of MDS MDS are marked by ineffective haematopoiesis and defective development of blood cells, e.g.: dyserythropoiesis (affects red blood cells production) dysgranulopoiesis (affects granulocytes production) dysmegakaryopoiesis (affects platelet production) Ineffective haematopoiesis and maturation of the blood cells results in one or more cytopenias, e.g.: anaemia (reduced red blood cell count) neutropenia (reduced absolute neutrophil count) thrombocytopenia (reduced number of platelets)

111 G-EXJ-1030713 May 2012 Minimal diagnostic criteria in MDS consensus, Vienna 2006 Prerequisite criteria constant cytopenia in one or more cell lineages complete blood count exclusion of all other causes of cytopenia / dysplasia MDS-related (decisive) criteria dysplasia in > 10% of all bone marrow cells in one or more of the lineages, or > 15% ringed sideroblasts complete blood count iron staining of bone marrow smears 519% blast cells bone marrow smears typical chromosomal abnormality karyotyping or FISH FISH = fluorescence in situ hybridization. Loken MR, et al. Leuk Res. 2008;32:5-17. Nimer SD. Blood. 2008;111:4841-51. Valent P, et al. Leuk Res. 2007;31:727-36.

112 G-EXJ-1030713 May 2012 Minimal diagnostic criteria in MDS consensus, Vienna 2006 (cont.) Additional criteria (for patients not fulfilling the decisive MDS criteria) abnormal phenotype of bone marrow cells flow cytometry molecular signs of a monoclonal cell population HUMARA assay, gene chip profiling, point mutation or SNP analysis markedly and persistently reduced colony formation CFU assay CFU = colony-forming unit; SNP-a = single-nucleotide polymorphism. Loken MR, et al. Leuk Res. 2008;32:5-17. Van de Loosdrecht AA. Leuk Res. 2008;32:205-7. Van de Loosdrecht AA, et al. Blood. 2008;111:1067-77. Van de Loosdrecht AA, et al. Haematologica. 2009;94:1124-1134. Nimer SD. Blood. 2008;111:4841-51. Valent P, et al. Leuk Res. 2007;31:727-36.

G-EXJ-1030713 May 2012 MDS classification and prognostic scoring

114 G-EXJ-1030713 May 2012 Diagnostic and prognostic of MDS Risk-stratification is necessary for clinical decision-making: predicts treatment outcomes predicts survival predicts risk of progression to AML Classification systemBasis of evaluation Derived prognostic score system FAB (1982)Cellular morphologyIPSS (1997) WHO (2002) Cellular morphology, cytogenetics WPSS (2007) FAB, French-American-British; IPSS, International Prognostic Scoring System; WPSS, WHO-based Prognostic Scoring System. Bennett JM, et al. Br J Haematol 1982;51:189199; Jaffe, et al, eds. Lyon: IARC Press; 2001; Greenberg P, et al. Blood 1997;89:20792088; Malcovati L, et al. J Clin Oncol 2007;23:35033510.

115 G-EXJ-1030713 May 2012 French-American-British (FAB) classification Blast percentage MDS Subtype Peripheral blasts (%) Bone marrow blasts (%) Additional features AML transformation (%) RARefractory anaemia140 Bennett JM, et al. Br J Haematol 1982;51:189199.

116 G-EXJ-1030713 May 2012 Survival by cytogenetic presentation in MDS patients Survival (%) 100 90 80 70 60 50 40 30 20 10 0 Time (years) 0123456789101112131415161718 Patients n(%) Y17(2) del(5q)48(6) Normal489(69) del(20q)16(2) Misc. single74(9) +838(5) Double29(3) Misc. double14(2) Chrom 7 abn.10(1) Misc. complex15(2) Complex66(8) Greenberg P, et al. Blood. 1998;89:2079-88.

117 G-EXJ-1030713 May 2012 Risk stratification and prognosis scoring for MDS: IPSS Influence of karyotype according to IPSS Good = normal, Y, del(5q), del(20q) Poor = complex ( 3 abnormalities) or chromosome 7 anomalies Int. = all other abnormalities Variable00.51.01.52.0 BM blasts (%)< 551011202130 KaryotypeGoodInt.Poor Cytopenia(s)0/12/3 Greenberg P, et al. Blood. 1998;89:2079-88. IPSS score

118 G-EXJ-1030713 May 2012 Years Cumulative survival of MDS patients by IPSS Low Int-1 Int-2 High 0246810141618 0 10 20 30 40 50 60 70 80 90 100 Survival Years Percent 0246810141618 0 10 20 30 40 50 60 70 80 90 100 Percent AML Evolution Low Int-1 Int-2 High Greenberg P, et al. Blood 1997;89:20792088. 12

119 G-EXJ-1030713 May 2012 MDS: WHO classification 2008 Blast percentage MDS Subtype Dysplasia Peripheral blasts (%) Bone marrow blasts (%) Ringed sideroblasts (%) Cytogenetics 5q syndromeMostly DysE< 1%< 5%< 15%5q sole RA, RN, RT, RCUDDysE, N, T< 1%< 5%< 15%Various RARSMostly DysE0< 5%> 15%Various RCMD23 lineagesrare< 5%< 15%Various RAEB-113 lineages< 5%59%< 15%Various RAEB-213 lineages519% Auer rods +/- 1019% Auer rods +/- < 15%Various MDS-U1 lineage< 1%< 5%< 15%Various BM = bone marrow; DysE = dyserythropoiesis; MDS-U = myelodysplastic syndrome, unclassified; N = neutropenia; pB = peripheral blood; RCMD = refractory cytopenia with multilineage dysplasia; RCUD = refractory cytopenia with unilineage dysplasia; RN = refractory neutropenia; RT = refractory thrombocytopenia; T = thrombocytopenia. Swerdlow SH, et al., eds. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. Lyon: IARC Press; 2008:109-38.

120 G-EXJ-1030713 May 2012 Survival of MDS patients according to transfusion dependency Transfusion-dependent patients Transfusion-independent patients Time (months) Proportion surviving 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 020406080100120140 Cazzola M, Malcovati L. N Engl J Med. 2005;352:536-8.

121 G-EXJ-1030713 May 2012 The WHO classification-based prognostic scoring system (WPSS) for MDS Points 0123 WHO subtype RA, RARS, del(5q) RCMD, RCMD-RS RAEB-1RAEB-2 Transfusion requirementNoneRegular Cytogenetic categoryGoodInt.Poor Risk groupsScoreMedian survival (months) Very low0103 Low172 Intermediate240 High3421 Very high5612 Malcovati L, et al. J Clin Oncol. 2007;25:3503-10. Transfusion dependence is an independent indicator of severity of disease and has a significant effect on survival

122 G-EXJ-1030713 May 2012 Overall survival and AML risk assessments in MDS by WPSS Malcovati L et al. J Clin Oncol 2007;25:35033510.

123 G-EXJ-1030713 May 2012 WHO prognosis scoring system allows time dependent prognosis scoring IPSSWPSS ProsMore widely used and recognized Allows time-dependent prognosis scoring and risk stratification Takes into account individual patient transfusion need ConsApplies only at the time of diagnosis Underestimates the impact of transfusion requirement and cytogenetics Underestimates the impact of poor cytogenetics Greenberg P, et al. Blood 1997;89:20792088. Malcovati L, et al. J Clin Oncol. 2007;25:35033510. Schanz J, et al. Blood. 2007;110:[abstract 248]. Kantarjian H, et al. Cancer. 2008;113:1351-61. Garcia-Manero G, et al. Leukemia. 2008;22:538-43.

G-EXJ-1030713 May 2012 Practical guide to bone marrow aspirate analysis in MDS

125 G-EXJ-1030713 May 2012 Bone marrow biopsy How is it done: Marrow aspirate and bone (trephine) biopsy are removed by physician in an outpatient procedure under local anesthesia heparinized sample can be stored at room temperature for 24 hours What tests are done? assessment of cellularity, architecture and focal collection of blasts for hematopoietic dysplasia smear staining examination cytogenetic analysis karyotype FISH What other tests might be done? flow cytometry cell counts cell sorting immunophenotyping Hellstrm-Lindberg E. Myelodysplastic Syndromes. London: Remedica; 2008. Van de Loosdrecht, et al, Haematologica.2009; 94:1124-34. Image from: Medline Plus. NIH/NLM. http://www.nlm.nih.gov/medlineplus/ency/imagepages/1129.htm. Accessed Jan, 2011.

126 G-EXJ-1030713 May 2012 Analysis of bone marrow smears Bone marrow aspirate smears are prepared on a slide by a medical technician using: Wrights stain Perls staining (for ringed sideroblasts) May-Grnwald-Giemsa staining Immunohistochemical stainings, i.e. CD34 A pathologist or hematologist then examines the slides for cell abnormalities under microscope: at least 400 nucleated cells and 20 megakaryocytes should be examined for morphology Hellstrm-Lindberg E. Myelodysplastic Syndromes. London: Remedica; 2008.

127 G-EXJ-1030713 May 2012 Abnormal large megakaryocyte (double arrow), abnormal hypo-granular and Pelger neutrophils (single arrows). Refractory anaemia (RA) Analysis of bone marrow smears (cont.) Refractory anaemia with excess blasts (RAEB) Erythroid cells with peri-nuclear iron accumulation Prussian blue staining (left); Perls stain showing ringed sideroblasts with peri-nuclear (mitochondrial) deposition of iron (right) ASH Image Bank, used with permission, all rights reserved. Courtesy of J. Goasguen, Universit de Rennes, France.

128 G-EXJ-1030713 May 2012 Immunophenotyping by flow cytometry Various lineages are labeled with antibodies that recognize specific haematopoeitic identifiers Combination of no more than four labels recommended Important for identification of blasts - CD34+/abnormal granularity/CD45dim Well-correlated with other diagnostic techniques and prognostic systems Van de Loosdrecht, et al. Haematologica.2009; 94:1124-34. Image from: http://www.bio.davidson.edu/courses/genomics/method/FACS.html

G-EXJ-1030713 May 2012 Cytogenetics of MDS in bone marrow aspirates

130 G-EXJ-1030713 May 2012 Importance of cytogenetic analysis in MDS Nearly half of the patients with MDS present with cytogenetic abnormalities 1 changes have a pathogenetic relevance (i.e. loss or gain of gene function) Cytogenetic analysis is essential for the diagnosis and classification of MDS according to IPSS and WPSS 2 Chromosomal aberrations have prognostic relevance for OS and for the time to leukaemic transformation, independent of other factors Cytogenetic analysis forms the basis for therapeutic decisions cytogenetics is indicative of response to therapy, i.e. lenalidomide in del(5q) 3 and azacitidine in 7/del(7q) 4 IPSS = International Prognostic Scoring System; OS = overall survival; WPSS = WHO classification-based Prognostic Scoring System. 1 Haase D, et al. Blood. 2007;110:4385-95. 2 Greenberg P, et al. Blood. 1998;89:2079-88. 3 List AF, et al. N Engl J Med. 2006;355:1456-65. 4 Fenaux P, et al. Lancet Oncol. 2009;10:223-32.

131 G-EXJ-1030713 May 2012 Cytogenetic aberrations are frequent in patients with MDS 9% 14% 29% 48% Normal karyotype One abnormality Two abnormalities Complex karyotype N = 2,072 patients with MDS Haase D, et al. Blood. 2007;110:4385-95.

132 G-EXJ-1030713 May 2012 WHO 2008: Incidence of the most common cytogenetic aberrations in MDS (over 5%) Unbalanced aberrations De novo MDS (%) Secondary MDS (%) +810 7/del(7q)1050 5/del(5q)1040 del(20q)5858 Y5 iso(17q)/t(17p)/del(17p)3535 Swerdlow SH, et al. In: WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. 4th ed. Lyon: IARC; 2008:441. Unbalanced aberrations with loss of genetic information (deletions or monosomies) are most common in MDS; balanced aberrations are rare

133 G-EXJ-1030713 May 2012 When should cytogenetic testing be performed in patients with MDS: Diagnosis WHO 2008 guidelines recommend a complete cytogenetic analysis of BM at initial diagnosis in all patients with MDS Cytogenetic analysis is mandatory for diagnosis of MDS associated with del(5q) patients with refractory cytopenia(s) who lack MDS diagnostic features; these patients may be considered as having presumptive evidence of MDS if they have MDS-related cytogenetic abnormalities (slides 4 and 5) rather than indicating abnormality, isolated loss of Y chromosome might be an age-related phenomenon and mosaicism with trisomy 8 might be a constitutional change. Therefore, they might not be sufficient to prove MDS BM = bone marrow. Swerdlow SH, et al. In: WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. 4th ed. Lyon: IARC; 2008:441. Valent P, Horny HP. Eur J Clin Invest. 2009;39:548-53. Vardiman JW, et al. Blood. 2009;114:937-51.

134 G-EXJ-1030713 May 2012 Is cytogenetic testing required for all patients? Yes, cytogenetic testing should be performed whenever possible at initial diagnosis and every 612 months during follow-up since karyotype and prognosis might change during the course of the disease. There are a few exceptions that will have no therapeutic consequences, e.g. very frail and multi-morbid patients. Are there disease presentations that correlate with specific cytogenetic abnormalities? There is no correlation between specific abnormalities and disease presentation (with exception of the association between isolated del(5q) syndrome and RA, with typical dysplasia of megakaryocytes). FAQs: Cytogenetic testing for diagnosis of patients with MDS Questions and answers were prepared under the review of Dr. Haase, University of Gttingen, Germany.

135 G-EXJ-1030713 May 2012 FAQs: Cytogenetic testing during treatment in patients with MDS Why is it important to perform cytogenetic testing during the treatment course? Cytogenetic remissions represent a better quality of remission and are more reliable than those determined by blood films or cytomorphology from the BM. Furthermore, karyotype might change during the course of the disease affecting the prognosis and the treatment of the patient. First data show that a cytogenetic progression might be detectable several weeks before clinical manifestation. Is cytogenetic testing important for all patients or is it recommended for specific focus groups? All patients with clonal abnormalities identified before start of therapy should be followed up during therapy. Also, patients with initially normal karyotype might develop abnormalities during the course of disease and, therefore, require regular monitoring. Questions and answers were prepared under the review of Dr. Haase, University of Gttingen, Germany.

136 G-EXJ-1030713 May 2012 Cytogenetics from BM is recommended when possible at least once every 6 months during therapy. When FISH is performed using peripheral blood (i.e. CD34-FISH), testing should be conducted every 3rd month. How often should cytogenetic testing be performed? When should treatment be altered as a result of changes in the cytogenetic profile? When a clear cytogenetic progression is seen, or when an abnormal clone is completely eliminated and karyotype turns normal and stays normal over time (at least 3 months); this depends on the type of therapy. What is the definition of cytogenetic progression in MDS? 1) Occurrence of new cytogenetic abnormalities (first in patients with normal karyotype or additional for patients with abnormalities). 2) Significant increase (> 50%) of the size of the clone with certain cytogenetic abnormalities. FAQs: Cytogenetic testing during treatment in patients with MDS (cont.) Questions and answers were prepared under the review of Dr. Haase, University of Gttingen, Germany.

137 G-EXJ-1030713 May 2012 Chromosome analysis in MDS: Karyotyping Each cell in the body contains 46 chromosomes representing the normal human karyotype MDS patients frequently have a clonal abnormality in the hematopoietic progenitor cells, where a proportion of these have an abnormal karyotype with altered number of chromosomes and/or large alterations in their structure Changes in the number of the chromosomes is due to monosomies (loss of a chromosome) and/or polysomies (more then 2 copies of a chromosome) Changes in the structure of the chromosomes are commonly noted as: deletions, when part or entire chromatid is missing insertions, when additional material is included in a chromosome translocations, when genetic material is exchanged between chromosomes

138 G-EXJ-1030713 May 2012 How to perform cytogenetic testing in patients with MDS: Karyotyping Sample Collect at least 1020 mL of heparinized BM aspirates Storage Samples should reach the lab within 24 hours after biopsy Number of metaphases needed Analyse 25 metaphases if possible, especially if the karyotype is normal, to exclude a small aberrant cell clone 678910 Metaphase quality To detect structural abnormalities, adequate chromosome banding is required ( 150250 bands per karyogram) See presenter for references.

139 G-EXJ-1030713 May 2012 How to perform cytogenetic testing in patients with MDS: Karyotyping (cont.) Medical technician prepares metaphase slides for karyotyping, by: culturing bone marrow aspirate for 24-72 hours, and then exposing the cells to slightly hypotonic solution synchronizing them in metaphase (e.g. using colchicine) finally, staining them (e.g. DAPI staining) and fixing them on slides A pathologist or hematologist then examines the slides for chromosomal abnormalities under microscope: Samples are examined under microscope manually or with aid of analysis software (separating, enhancing banding pattern, chromosome pairing) At least 25 metaphases should be examined, especially if the karyotype is normal, to exclude a small aberrant cell clone Holland and Frei. Cancer Medicine 6. American Cancer Society; 2003. Lucia Cytogenetics. http://www.lucia.cz/en/products/lucia-karyo. Accessed Feb.2011.

140 G-EXJ-1030713 May 2012 Can peripheral blood be used? Peripheral blood is usually not an alternative. No BM available: attempt banding analysis from peripheral blood (CD34-FISH), especially when blasts are present. Metaphase yield from peripheral blood is generally worse than that from BM aspirates. CD34-FISH can be an option. 1 What sample-related factors could influence the accuracy of the test? Ex-vivo time over 24 hours: clotting; bacterial/fungal contamination; and low cellular content (e.g. hypocellular BM, or if several syringes are filled during biopsy and the last syringe is used for banding analysis). Need for dividing cells; cell clones < 10% of abnormal cells can be overlooked; submicroscopic abnormalities cannot be detected. What are the limitations of this method? FISH = fluorescent in situ hybridization. 1 Braulke F, et al. Leuk Res. 2010;34:1296-301. Questions and answers were prepared under the review of Dr. Haase, University of Gttingen, Germany. FAQs: Karyotyping

141 G-EXJ-1030713 May 2012 Chromosome analysis in MDS: Fluorescence in situ hybridization (FISH) Fluorescently labeled DNA probes recognize complementary sequences on the chromosomes probes that recognize centromeres detect changes in the number of chromosomes probes that recognize specific genes can detect changes in the chromosome sequence Fluorescently labeled probes for bcr and abl genes show exchange of DNA between Chromosomes in chronic myelogenous leukemia Images from: http://www.wikidoc.org/index.php/Chronic_myelogenous_leukemia. Accessed Feb.2011.

142 G-EXJ-1030713 May 2012 Preparation of the FISH sample Metaphase slides for karyotyping could also use bone marrow smears and paraffin-embedded bone marrow biopsies) Denature DNA (heat up the sample) Hybridize with denatured (pre-heated) labeled probes Wash to remove excess probe DAPI stain for DNA View with fluorescence microscope and take photographs Image from: http://www.creative-biolabs.com/fish/tissuearray5.htm. Accessed Feb. 2011 Protocol from: labs.mmg.pitt.edu/gjoerup/FISH%20protocol%20Vysis.doc. Accessed Feb. 2011. Preparation of the FISH could be done by trained medical technician or clinical geneticist. The analysis of the samples is carried by clinical geneticists and/or hematologists.

143 G-EXJ-1030713 May 2012 FISH: Selection of probes MDS FISH panel for initial diagnosis detects the most common aberrations, and typically includes probes for 5q, 7q, #8, 11q, 12p, 13q, 17p, and 20q Probes*ManufacturerWeb site Multiprobe MDS/AML panelCytocellwww.cytocell.com 5/5q, 7/7q, 8cen, 20q Genzyme Genetics www.genzymegenetics.com 5p/q, 7q, 17p13, 20q13Kreatechwww.kreatech.com 5p/q, 7cen/q, 8cen, 17p13, 20q13, Ycen Abbott Molecular www.abbottmolecular.com 5/5q, 7/7q, 20qMetasystems www.metasystems- international.com Selection of the FISH probes is essential part of the analysis and is typically done by clinical geneticists in cooperation with hematologists *Examples are not representative of the complete spectrum of probes available from each provider. 1 Cherry AM, et al. Blood. 2010;116:[abstract 2922].

144 G-EXJ-1030713 May 2012 How is a probe selected? Use the standard MDS FISH panel for initial diagnosis. When the aberration is known/suspected, use additional probes in the region. If disease morphology is suggestive of a certain genotype, one could directly use the respective probe e.g. if RA with typical dysplasia of megakaryocytes occurs, then use probes for del(5q). Note, however, that one can miss other genetic changes with this approach. How many probes are typically used in a panel? A standard panel consists of 78 probes. 1 An extended panel can include up to 12 probes. FAQs: FISH 1 Cherry AM, et al. Blood. 2010;116:[abstract 2922]. Questions and answers were prepared under the review of Dr. Haase, University of Gttingen, Germany.

145 G-EXJ-1030713 May 2012 The available survival data are based on karyotyping (banding) studies. However, in addition to karyotyping, one should use a standard FISH panel: 5p/q, 7cen/q, #8cen, 17p13, 20q13, and Ycen (in males). What probes should be used for survival prognosis? What are the limitations of FISH? One sees/finds what one is looking for; therefore, one could overlook complex or rare abnormalities (e.g. finding an isolated del(5q) by FISH might give a false good prognosis if it is a part of a complex genotype that has been misidentified). FAQs: FISH (cont.) Questions and answers were prepared under the review of Dr. Haase, University of Gttingen, Germany.

147 G-EXJ-1030713 May 2012 Summary WHO 2008 guidelines recommend complete cytogenetic analysis of BM at initial diagnosis of MDS Chromosomal aberrations, among other factors, have prognostic relevance on overall survival and time to leukaemic transformation IPSS was first to define cytogenetic risk groups and to show an association with the survival prognosis of the patient improved understanding of the cytogenetic risk factors provides a better prognosis scoring for the patients with MDS Approximately 50% of MDS patients have abnormal cytogenetics in addition, it is supposed that many patients with normal cytogenetics actually have clonal abnormalities that remain undetected by metaphase cytogenetics

148 G-EXJ-1030713 May 2012 Summary (cont.) Cytogenetic analysis is essential for making therapeutic decisions with regard to patients with MDS as it has shown associations with the response to hypomethylating and immunomodulating agents Sequential cytogenetic analysis is recommended to improve clinical management in MDS

G-EXJ-1030713 May 2012 TRANSCRANIAL DOPPLER ULTRASONOGRAPHY (TCD) FOR ASSESSMENT OF STROKE RISK IN SICKLE CELL DISEASE NOTE: These slides are for use in educational oral presentations only. If any published figures/tables from these slides are to be used for another purpose (e.g. in printed materials), it is the individuals responsibility to apply for the relevant permission. Specific local use requires local approval.

150 G-EXJ-1030713 May 2012 Outline Sickle cell disease (SCD) Transcranial Doppler (TCD) TCD in SCD TCD Equipment Guidelines for TCD in SCD Summary LIC = liver iron concentration; MRI = magnetic resonance imaging; SF = serum ferritin; SIR = signal intensity ratio; SQUID = superconducting quantum interface device.

G-EXJ-1030713 May 2012 Sickle Cell Disease (SCD)

152 G-EXJ-1030713 May 2012 What is SCD? An inherited disorder affecting haemoglobin (Hb) synthesis Sickle cell erythrocytes have a mutant form of Hb and HbS resulting from GluVal mutation in 6th codon of -globin chain HbS turns normally pliable erythrocytes into rigid, sickle-shaped cells The irregular erythrocyte morphology leads to episodes of vascular occlusion and acute pain progressive organ damage Children have increased risk of infection and stroke Life expectancy may be shortened HbS = sickle cell haemoglobin. Schnog JB, et al. Neth J Med. 2004;62:364-74. Image from www2.med.umich.edu/prmc/media/newsroom/downloadImages.cfm?ID=656.

153 G-EXJ-1030713 May 2012 Clinical manifestations of SCD AnaemiaRed cell survival ~ 17 days (120 days in healthy people) 1 PainAcute and chronic 1 Central nervous systemOvert stroke, silent stroke, and neurocognitive impairment 13 Pulmonary Recurrent acute chest syndrome, pulmonary hypertension, and chronic sickle lung disease 1,2 SkinChronic ulcers, typically around the ankles 1 Joints Osteonecrosis (avascular necrosis) of femoral and humeral heads 1,2 EyesRetinal ischaemia, detachments sickle retinopathy 1,2 Kidneys Inability to concentrate urine; proteinuria progressing to nephrotic syndrome; end-stage renal failure 4 CardiovascularCardiac decompensation and cardiomyopathy 1 1 Schnog JB, et al. Neth J Med. 2004;62:364-74. 2 Claster S, Vichinsky EP. Br Med J. 2003;327:1151-5. 3 Prengler M, et al. Ann Neurol. 2002;51:543-52. 4 Ataga KI, Orringer EP. Am J Hematol. 2000;63:205-11.

154 G-EXJ-1030713 May 2012 Prevalence of SCD The frequency of the HbS gene is highest in populations in which malaria is (or was) endemic 1,2 Approximately 200,000 new cases of SCD occur in Africa every year 1 Recent population migrations have led to an increase in disease frequency in other areas 1 Weatherall DJ, Clegg JB. Bull World Health Organ. 2001;79:704-12. 2 Modell B, Darlison M. Bull World Health Organ. 2008;86:480-7. 3 Sickle cell disease: screening, diagnosis, management, and counseling in newborns and infants clinical practice guideline number 6 AHCPR 1993;Publication 93-0562. 4 Sickle Cell Society. Sickle Cell Society Publication SC4 2005: www.sicklecellsociety.org/pdf/SC4.pdf. Image from Christianson A, et al. March of Dimes Global Report on Birth Defects: the hidden toll of dying and disabled children. 2006 (www.marchofdimes.com/MOD-Report- PF.pdf). 1 out of 2,400 live births (all ethnic groups) are affected in England, where 12,500 individuals live with the disease 4 Births with a pathological Hb disorder per 1,000 live births

165 G-EXJ-1030713 May 2012 Correlation between TAMMV on TCD and stenoses on MRA in SCD Study rationale relationship between neuroimaging abnormalities and TCD is unclear in adult patients with SCD imaging abnormalities reported in up to 44% of children with SCD; prevalence in adults unknown Differences: adults vs children frequency of imaging abnormalities in a

G-EXJ-1030713 May 2012 DIAGNOSTICS SLIDE DECK. 2 G-EXJ-1030713 May 2012 TABLE OF CONTENTS ● Use of...

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