QUANTITATIVE IMAGING OF HUMAN LIVER IRON CONCENTRATIONS IN VIVO Tim St Pierre1, A Fleming1, W Chua-anusorn1, P Clark1, E Rossi, G Jeffrey2,3, J Olynyk2 The vision we will be presenting to you today is the development of an internet service industry for the assessment of iron overload by MRI, which has emerged as an exciting possibility from our research into body-iron overload, and from our development of a novel imaging methodology which enables the generation of [Fe] maps. And we will outline a time-frame over which this could be developed into a multi-million dollar service industry.

Declaration of competing interests Three of the investigators on this project are involved with a commercial venture based on non-invasive measurement of liver iron concentrations Tim St Pierre Paul Clark Wanida Chua-anusorn

Standard methods of assessing the body iron burden Ferritin serum assay Needle biopsy of the liver So let’s look at the ...

Liver biopsy

Non-invasive measurement of liver iron concentration

The magnetic resonance imager (MRI)

A liver full of magnets!

Proton transverse relaxation rate (R2) image and distribution … You can see the map here, which has been superimposed on one of the five T2-weighted images acquired for the calculations. Now, the power of this development lies not only in the ability to see the heterogeneity of iron loading throughout the liver, the degree of which can be parameterised by the width of the Gaussian distribution of the relaxivity values, but by the position of the peak, which quantifies the mean level of iron loading in the liver. If we go to the next slide … Clark et al, Mag Res Imaging 18 (2000) 431-438.

Liver R2 images and distributions healthy volunteer 3 iron loaded subjects with sequentially increasing liver iron concentrations

R2 image analysis of human liver samples Iron loaded liver tissue: dissection outline

R2 image analysis of human liver samples Clark et al, Magn Reson Med 49 (2003) 572-575

R2 image analysis of human liver samples Clark et al, Magn Reson Med 49 (2003) 572-575 Mean R2 vs iron concentration for 32 cubes of liver dissected from a single iron loaded liver post mortem.

Measurement of liver R2 in vivo Needle biopsy samples a few milligrams of tissue from the right hand side of the liver

Relationship between <R2> in right hand side of liver and needle biopsy iron concentration (dry wt) St Pierre et al (2005) Blood 105, 855-861

Sensitivity and specificity of R2-LIC measurements to biopsy LIC measurement LIC Threshold mg Fe/g dry Clinical Relevance Sensitivity Specificity 1.8 Upper 95% of normal 94% (86-97) 100% (88-100) 3.2 Suggested lower limit of optimal range for liver iron concentrations for chelation therapy in transfusional Fe overload * (85-98) (91-100) 7.0 Suggested upper limit of optimal range for liver iron concentrations for transfusional Fe overload and threshold for increased risk of iron induced complications * 89% (79-95) 96% (86-99) 15.0 Threshold for greatly increased risk for cardiac disease and early death in patients with transfusional iron overload * 85% (70-94) 92% (83-96) * Olivieri NF, Brittenham GM. Blood. 1997; 89: 739-761

Measurement of R2 standards

Measurement of R2 standards St Pierre et al (2005) Blood 105, 855-861 R2 vs paramagnetic Mn2+ concentration for the same series of phantoms measured on 13 different 1.5T MRI scanners

Reproducibility of liver R2 measurements on 2 MR scanners Random error: 7.7% Systematic error: 1.2% St Pierre et al, NMR in Biomed 2004; 17, 446-458

Assessment of liver damage risk from iron loading

Using age and LIC at diagnosis to predict fibrosis grade in HH Olynyk et al (2005) Am. J. Gastroenterol 100, 837

Using age and LIC at diagnosis to predict fibrosis grade in HH Olynyk et al (2005) Am. J. Gastroenterol 100, 837

Relationship of R2 distribution with liver histology 250 mm Perl’s Stain 250 mm Reticulin Stain Clark et al, Magn Reson Med 49, (2003) 572-575

Relationship of R2 distribution with liver histology 250 mm Perl’s Stain 250 mm Reticulin Stain Clark et al, Magn Reson Med 49, (2003) 572-575

Relationship of R2 distribution with liver histology 250 mm Perl’s Stain 250 mm Reticulin Stain Clark et al, Magn Reson Med 49, (2003) 572-575

R2 distribution and liver biopsy histology Non-cirrhosis LIC = 10.2 mg/g DW R2 = 99 ± 20 Cirrhosis LIC = 12.6 mg/g DW R2 = 132 ± 50

R2 distribution and liver biopsy histology Mild Fibrosis LIC = 1.1 mg/g DW R2 = 30.9 ± 7.4 The possibility also exists that R2-MRI may be developed to identify cirrhosis in patients with normal levels of liver iron. Note that the HIC is in the normal range. Cirrhosis LIC = 1.8 mg/g DW R2 = 29.9 ± 13.4

Relationship between LIC and total body iron stores in hereditary hemochromatosis

Serum ferritin and body iron stores in hereditary hemochromatosis Olynyk et al (1998) Am. J. Gastroenterol. 93, 346

LIC by biopsy vs total body iron stores by quantitative venesection Hereditary Hemochromatosis Thalassemia Angelucci et al (2000) N Eng J Med 343, 327 Summers et al (1990) Hepatology 12, 20 Olynyk et al (1998) Am. J. Gastroenterol. 93, 346

Body iron store distribution Liver iron store Extrahepatic iron store Liver volume V Total body iron store = liver iron store + extrahepatic iron store Liver iron store = liver iron concentration x liver volume

Measuring total body iron stores (TBIS) and liver iron stores Measure LIC 3 times during venesection Measure liver volume Weighted fit through data Extrapolate LIC to zero to obtain TBIS Extrapolate LIC to 1 mg Fe/g dw for comparison of TBIS with other studies

Subjects & methods Newly diagnosed hereditary hemochromatosis subjects were recruited (n=19) (male = 12, female = 7) 17 C282Y homozygotes, 1 C282Y/H63D, 1 Wild-type/H63D LIC measured with R2-MRI [6] Liver volume measured with MRI simultaneously Subjects recalled for follow-up LIC measurements at estimated half way point in venesection schedule and near end of schedule

Non-invasive monitoring of LIC during venesection Subject 1 Subject 2 Subject 3 Subject 4 LIC measured using R2-MRI. Dashed line is upper limit normal Subject 5 Subject 6 Subject 7 Solid lines are weighted fits to the data yielding estimate for initial LIC and total body iron store (from mL of blood to reach zero LIC).

LIC vs TBIS by quantitative venesection in HH Biopsy Measurements R2-MRI Measurements Summers et al (1990) Hepatology 12, 20 Olynyk et al (1998) Am. J. Gastroenterol. 93, 346 1.65 1.09 Gradients

LIC vs TBIS by quantitative venesection Biopsy Measurements (thalassemia) R2-MRI Measurements (hereditary hemochromatosis) Angelucci et al (2000) N Eng J Med 343, 327

LIC vs TBIS by quantitative venesection Biopsy Measurements (thalassemia) R2-MRI Measurements (hereditary hemochromatosis) Gradient 7.3 10.6 Angelucci et al (2000) N Eng J Med 343, 327

Body iron store distribution Liver iron store Extrahepatic iron store Liver volume V Total body iron store = liver iron store + extrahepatic iron store Liver iron store = liver iron concentration x liver volume

a is fraction of total body iron store in the liver Liver iron store Extrahepatic iron store Liver volume V Total body iron store = liver iron store / a

Fraction (a) of iron store in liver Mean fraction of iron stores in liver is 45% Range 26 - 80%

New model for iron distribution in HH Assume fraction,  , of total body iron store in the liver varies linearly with rate of liver iron loading (LIC/Age)  0 LIC / Age Gradient = 

New model for predicting iron stores in HH 95% limits of agreement New Model: ± 40% Using LIC: ± 72% Using Ftn: ± 102%

Summary (1) R2 imaging …… can be used to measure non-invasively liver iron concentrations (LIC) with known accuracy and precision has good sensitivity and specificity for measurement of LIC at both low and high LIC ranges has dynamic range of measurement from normal LIC to the very highest concentrations encountered in clinical practice works on most 1.5 T MRI units has health regulatory authority clearance for LIC measurement in USA (FDA), Europe (CE Mark), and Australia (TGA) may have the potential to detect liver cirrhosis/fibrosis

Summary (2) Non-invasive measurement of LIC …… Enables serial monitoring of patients on blood transfusion and chelation therapy to aid in chelation dose determination Aids in the identification of newly diagnosed hemochromatosis patients who are at risk of iron induced liver damage

Summary (3) Non-invasive measurement of LIC and liver volume… Has demonstrated a correlation between fraction of TBIS in the liver and rate of iron loading in HH Enables a more accurate prediction of venesection requirements for HH subjects compared with LIC or serum ferritin measurements

Co-workers School of Physics, University of Western Australia Paul Clark Wanida Chua-anusorn Adam Fleming School of Medicine, University of Western Australia Gary Jeffrey John Olynyk Ric Rossi Thalassemia Research Center, Mahidol University Pensri Pootrakul Department of Haematology, Prince of Wales Hospital Rob Lindeman SKG Radiology Erin Robins