Measuring*liver*iron*contentin* …...Heterogeneity*of*iron*concentraon* throughoutthe*liver* Sample...
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Measuring liver iron content in thalassaemia and sickle cell disease Maciej Garbowski, MD, University College London
Transcript of Measuring*liver*iron*contentin* …...Heterogeneity*of*iron*concentraon* throughoutthe*liver* Sample...
Measuring liver iron content in thalassaemia and sickle cell disease
Maciej Garbowski, MD, University College London
What is liver iron? • Cellular FerriAn (not serum ferriAn) • Haemosiderin (old ferriAn, parAally degraded iron rich) • Labile iron pool (regulatory) • Iron in cellular enzymes, as prostheAc groups: heme, iron-‐sulfur cluster
– Mitochondria • <1.8mg/gdw normal range LIC (liver iron content)
Where is liver iron? • Hepatocyte (parenchymal): typically from gut
uptake of diet iron, also during redistribuAon from spleen – HFE haemochromatosis – Thalassaemia intermedia:
• As transferrin iron uptake±NTBI uptake • Hb-‐Haptoglobin uptake • Haem-‐haemopexin, methaemalbumin uptake
• Macrophage (Kupfer cells) – Transfusional haemosiderosis
• Hb-‐haptoglobin • As red cells aXere splenectomy BaYs K. Mod Pathol 2007
Why measure liver iron overload? • LIC predicts total body storage iron in TM1
• Absence of pathology – heterozygotes of HH where liver levels < 7 mg/g dry weight
• Liver pathology – abnormal ALT if LIC > 17 mg/g dry weight2 – liver fibrosis progression if LIC > 16 mg/g dry weight3
• Cardiac pathology at high levels
– Increased LIC linked to risk of cardiac iron in unchelated paAents 2,6
– LIC >15 mg/g dry weight associaAon with cardiac death • all of 15/53 TM paAents who died4 • improvement of subclinical cardiac dysfuncAon with venesecAon alone post-‐BMT5
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. BriYenham GM, et al. N Engl J Med. 1994;331:567-73. 5. Mariog E, et al. Br J Haematol. 1998;103:916-‐21. 6. Buja LM, Roberts WC. Am J Med. 1971;51:209-‐21
ALT = alanine aminotransferase; BMT = bone marrow transplantaAon.
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Body iron (mg/kg) = 10.6× hepaAc iron concentraAon (mg/g dry weight)
Sample <1 mg Dry Weight (n=23)
Body iron
stores (m
g/kg)
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0
r=0.83
0 5 10 15 20 25 HepaAc iron concentraAon (mg/g dry weight)
Angelucci et al. N Engl J Med. 2000;343:327.
Liver Iron ConcentraAon Predicts Total Body Iron Stores
0 5 10 15 20 25
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r=0.98
Body iron
stores (m
g/kg)
HepaAc iron concentraAon (mg/g dry weight)
Sample >1 mg Dry Weight (n=25)
Liver enzymes and iron overload
AST U/L
16.8mg/g dry wt
AL
T U/L
Jensen PD, et al. Blood. 2003;101:91-‐6
How to measure liver iron
• IntervenAonal – Biopsy
• Non-‐intervenAonal – SQUID – MRI – Serum markers?
Biopsy-‐measured LIC • Standard procedure 16 g needle under local anesthesia
typically into right lateral lobe • Small sample about 1g dry weight, up to 4g wet weight • Fresh/Fixed (PFA) • Allows for histology assessment, staging, grading • Semi-‐quanAtaAve Perl stain for iron and hepatocyte vs
Kupfer cell distribuAon • QuanAtaAve biochemical analysis of iron content per gram
Assue: – Dry weight: aXer drying – Wet weight – at biopsy before fixing with PFA or unfixed – Paraffin embedded for histology:
• Dewaxing causes fat deposits to be washed away with solvents
• Tissue weight is reduced, denominator is lower, LIC higher
2 cm
LIC by biopsy: Disadvantages • Coefficient of variability 19-‐40%
– Due to patchy iron distribuAon in the liver (distribuAon artefact)
– Small sample size – Effect of fibrosis
• no global LIC esAmaAon • Not standardized – colorimetric vs Atomic AbsorpAon • Wet/dry raAo in different labs • ComplicaAons • PaAent preference • Rarely used in chelaAon clinical trials
Heterogeneity of iron concentraAon throughout the liver
Sample size and type CV of LIC Pathology Source
Needle biopsy (< 4 mg dry weight)
19% Normal Emond, et al. 1999 Kreeftenberg, et al. 1984
Needle biopsy
(< 4 mg dry weight) > 40% End-stage
liver disease Emond, et al. 1999 Kreeftenberg, et al. 1984
Needle biopsy (9 mg dry weight)
9% Normal Barry, Sherlock. 1971
“Cubes” (200–300 mg wet weight)
17% 24%
β-thalassaemia
Non-cirrhotic
Ambu, et al. 1995
“Cubes” (1,000–3,000 mg wet weight),
19% β-thalassaemia Part-cirrhotic
Clark, et al. 2003
CV = coefficient of variaAon.
Ambu R, et al. J Hepatol. 1995;23:544-‐9; Barry M, Sherlock S. Lancet. 1971;1:100-‐3;
Clark PR, et al. Magn Reson Med. 2003;49:572-‐5; Emond MJ, et al. Clin Chem. 1999;45:340-‐6;
KreeXenberg HG, et al. Clin Chim Acta. 1984;144:255-‐62.
How to measure liver iron
• IntervenAonal – Biopsy
• Non-‐intervenAonal – Serum markers? – SQUID – MRI
Use serum ferriAn instead of LIC ? • Advantages
• Simple • Widely available • Serum ferriAn broadly correlated with body iron (macrophages) • Validated as predictor of complicaAons of iron overload in TM
• Disadvantages • Origin of serum ferriAn differs above values of 4K • Raised by inflammaAon or Assue damage (e.g. sickling crisis) • Lowered by vitamin C deficiency • RelaAonship of ferriAn to body iron varies in different diseases
• Low relaAve to LIC in Thal intermedia (hepatocellular > macrophages) • Higher and variable in SCD
Non-‐intervenAonal LIC measurement
• Serum markers: FerriAn – RelaAonship with LIC is different in SCD, TI and TDT
– Hepatocyte iron overload has disproporAonately low serum ferriAn unAl liver damage occurs and Assue ferriAn is released
– Kupfer cell and RES: transfusional iron overload is beYer marked by secreted ferriAn Origa, Hamatologica 2007, 92 583
How to measure liver iron
• IntervenAonal – Biopsy
• Non-‐intervenAonal – Serum markers? – SQUID=superconducAng quantum interference device
– MRI • R2* i.e. 1/T2* Anderson 2001 proof of concept • R2 i.e. 1/T2 (Ferriscan) St Pierre 2005
Principle of MR imaging for iron
• Iron overload shortens the Assue relaxaAon Ames in the longitudinal axis (T1) and transverse axis (T2) on which magneAc resonance imaging is based
• The decrease in the intensity of spin echo images (1/T2 or R2) with iron overload derives from shortening in the T2 relaxaAon Ames (Leung et al., 1984; Stark, 1991).
• This T2 shortening is mainly due to the paramagneAc properAes of ferriAn iron (Brown et al., 1985) (Stark, 1991)
• The spin echo can be detected in several ways, the shorter the echo Ame, the greater the sensiAvity
• The gradient echo relies on mulAple echos over a shorter acquisiAon Ame period than spin echo techniques
R2* = R2 + R2’
1/T2* = 1/T2 + 1/T2’
Intrinsic effects Tissue relaxaFon
Extrinsic effects MagneFc inhomogeneity
R2*
[Fe]
[Fe]
T2*
• Gradient echo (T2*) – Long first echo Ames (2-‐20ms) – not suitable for high LIC – MulAple breath-‐holds – Variable weights of biopsy specimens – BeYer calibraAon with non-‐fibroAc samples
• Normal liver T2* 33±7ms • LIC=0.0146(R2*)-‐0.45; T2* of 5ms=2.47mgFe/g dw • Main purpose to establish the relaAonship between Assue iron and T2* rather than exact quanAficaAon of iron
T2*LIC by Anderson et al.
Anderson L, et al. Eur Heart J 2001;22:2171-‐9.
T2*LIC by Anderson et al. • paper focused on cardiac iron • but this was the 1st report to show Proof of Concept that
Assue iron relates to Assue MR relaxivity (T2*)
• Cardiac T2* not related to heart Assue iron, • this relaAonship shown for liver iron in 30
paAents with beta thalassaemia, therefore • in general: Assue T2* relates to Assue iron
more iron
Anderson L, et al. Eur Heart J 2001;22:2171-‐9.
T2* RBH-‐UCLH • Updated T2* LIC method, used at UCLH, Heart Hospital, Bart’s and RBH – 50 liver biopsies from 25 paAents paired with MRI scan
– Newer T2* acquisiAon: shorter first echo Ame, more echo Ames
– Single laboratory biopsies as part of deferasirox registraAon studies (Renne, France)
Garbowski et al 2014, JCMR 16(1) p.40
T2*LIC RBH-‐UCLH • 1.5T Sonata MR scanner at Royal Brompton Hospital, London.
• Transverse slice through the centre of the liver using a mulA-‐echo single breath-‐hold gradient echo T2* sequence -‐ echo Ames TE 0.93-‐16.0ms, shorter and more closely spaced
• T2* decay was measured using Thalassaemia tools (Cardiovascular Imaging SoluAons, London, UK) from a region of interest (ROI) in an area of homogeneous liver Assue, avoiding blood vessels.
• To account for background noise, a truncaAon method was used for curve-‐figng.
• All T2* measurements were performed in triplicate by 2 independent observers choosing three separate ROIs to analyse.
• The Regions of Interest were chosen to be as large as possible in three separate areas (anterior, mid/lateral and posterior).
• Corrects Anderson T2*LIC by 220%: T2*=5 ms is now 2.47*2.2=5.43mgFe/g dry weight
Method
Garbowski et al 2014, JCMR 16(1) p.40
Other T2*LIC callibraAons
Wood JC, et al. Blood. 2005;106:1460-‐5.
HIC = hepaAc iron concentraAon.
Hankins JS, et al. Blood. 2009;113:4853-‐5.
1/T2*=R2*
Comparing T2*-‐LIC calibraiAons
Garbowski et al 2014, JCMR 16(1) p.40
1/T2*=R2*
R2-‐LIC Ferriscan • Transverse images with a mulA-‐slice single spin-‐echo (SSE) pulse
sequence • Slice thickness of 5 mm • 25 minute acquisiAon • Frequently employed in chelator clinical trials • Central reporAng in Perth, Australia • Validated regularly for inter-‐site reproducibility
St Pierre TG, et al. Blood. 2005;105:855-‐61
No iron overload
Thalassemia major
R2 (s-1)
R2 (s-1)
233
194
155
116
77
0
0 80 160 240 320 400
0 80 160 240 320 400
Voxels 500
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R2-‐LIC Ferriscan
St Pierre TG, et al. Blood. 2005;105:855-‐61
24 St Pierre et al. Blood. 2005;105:855.
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Biopsy iron concentraAon (mg/g dry Assue)
Mean transverse re
laxaAo
n rate R
2 (s-‐1)
HepaAAs
Hereditary hemochromatosis
Beta-‐thalassemia/hemoglobin E
Beta-‐thalassemia
St Pierre TG, et al. Blood. 2005;105:855-‐61.
RelaAonship between R2 and needle biopsy LIC (dry weight)
Comparing Ferriscan and T2*LIC • 92 scans in 54 paAents with transfusional
iron overload • Bland-‐Altman analysis showed
unacceptably wide limits of agreement • THEREFORE methods cannot be used
interchangeably when following LIC trends • One method should be used to follow
trends
Garbowski et al 2014, JCMR 16(1) p.40
Non-‐interchangeability of R2LIC and T2*LIC
• Poor agreement between T2* LIC and R2LIC – not derived from ROI-‐related and reproducibility-‐related variability of
both methods • T2*LIC and Ferriscan have excellent reproducibility
– likely stems from different sensiAvity of R2 and R2* to iron distribuAon in the Assue and to sources of noise.
– Further studies necessary
Case 1 • 30 yo TD Eβ-‐Thalassaemia paAent on DFX 30mg/kg/d, variable compliance historically, on
clinical trial with sotatercept (reported 2014 ASH abstract) • T2*LIC Jan 2015 25mg/gdw, no cardiac iron • Serum FerriAn trend 3000 down to 1400ug/L on DFX but drop in transfusion requirements
by 33%: ILR reduced from 0.32mgFe/kg/d to 0.22mgFe/kg/d • “SoX landing” with DFX as ferriAn approaching 1000ug/L – dose was to be reduced from 30
to 20mg/kg • Ferriscan was requested Oct 2015
– R2-‐LIC 23mg/gdw
• Is the LIC reduced? – Difficult to tell. SF trend suggests yes
• Should DFX dose be reduced? – But what about LIC ?
next management step?
Request T2*LIC to compare with baseline Ferriscan and T2*LIC don’t agree – poor agreement on Bland-‐Altman in the same paAent Longitudinal trends in LIC should be read using the same LIC method
S-‐ferriAn [ug/L]
ALT [IU
/L]
LIC=25 mg/g 23 mg/g
Case 2 • 28 yo TDT paAent on 25mg/kg/d DFX
approaching the “soX-‐landing” threshold for dose reducAon
• 3u PRBC/month ILR=0.4mgFe/kg/d • 16 Nov 2014 T2* LIC 4mg/gdw with SF
~1500ug/L, no cardiac iron • Develops acute kidney injury in Apr 2015
at SF 1100ug/L • DFX was stopped • S-‐creaAnine checked weekly,
normalized by Jul 2015 • Off-‐treatment 6 weeks • FerriAn increased>2000ug/L
• Restarted at DFX 0.5g/OD (10mg/kg/d) • Sep 2015 R2LIC 3.8mg/gdw and T2*LIC
6.0mg/gdw • LIKELY reached normal liver iron despite
SF>1000 when AKI occurred in Apr 2015
S-‐ferriAn [ug/L]
S-‐crea [u
mol/L]
LIC=4 mg/g 6 mg/g ??
FerriAn LIC?
• There is a disconnect between LIC and ferriAn in some paAents which may lead to problems with AtraAng chelator dose.
LIC and serum ferriAn use in chelaAon studies
• The primary outcome in establishing efficacy is typically LIC difference and typically measured by R2LIC (Ferriscan) or R2*LIC
• FerriAn trend is similarly useful • However absolute ferriAn value less so • FerriAn response vs LIC response problem (317paAents on DFX>1year)
– Serum ferriAn response • Occurs in 73% of paAents • Predicts LIC response in 80% of paAents • Is more likely to predict LIC response when baseline serum ferriAn is <4000 ng/mL
(88 vs 70%) • Closer correlaAon of ferriAn with LIC trends when ferriAn <4000ng/ml
– Serum ferriAn nonresponse • Occurs in 27% of paAents • Over half of these (52%) have a LIC response • Is more likely with
– Higher transfusional iron intake – Lower deferasirox dose
Porter et al 2014 ASH oral presentaAon 2014
Serum ferriAn response predicts an LIC response in more paAents when baseline serum ferriAn is <4000 ng/mL
88.7
52.6
70.3
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ents (%
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Serum ferriAn nonresponders show a similar LIC response rate irrespecAve of baseline serum ferriAn
Serum ferriFn nonresponders
Serum ferriFn responders
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Baseline serum ferriFn <4000 ≥4000 <4000 ≥4000
Porter et al 2014 ASH oral presentaAon 2014
Stronger correlaAon between serum ferriAn and LIC absolute change when baseline serum ferriAn <4000 ng/mL
LIC absolute change from
baseline
(mg Fe/g dw)
20
-‐30
-‐20
-‐10
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Serum ferriAn absolute change from baseline (ng/mL)
-‐12,000 8000 -‐8000 -‐4000 0 4000
32 Porter et al 2014 ASH oral presentaAon 2014
Conclusions • Tissue ferriAn and haemosiderin iron shortens MR relaxiviAes in the
Assue thus allowing for iron quanAficaAon by MRI • Biopsy based quanAficaAon of LIC is being replaced by MRI
methods • Ferriscan R2LIC is well established clinically and in research, longer
acquisiAon, reports available within a few days (Australia) • T2*LIC has been improved, acquisiAon is rapid and can be used
together with heart T2*, reports may be on the same day. • T2*LIC and Ferriscan R2LIC are not interchangeable – one method
needs to be used in following up trends • Serum ferriAn trend very useful and typically follows LIC trends but
there are discrepancies when ferriAn >4000ug/L • FerriAn-‐LIC relaAonship differs in SCD, TDT and NTDT
Thank you
QuesAons?
SQUID (liver susceptiometry)
• First validated non-invasive method for liver iron • Linear relationship to iron by biopsy • Only 4 operating machines in world • Expensive but room temperature devices being developed • Unclear about comparison between centres • Underestimated LIC in deferasirox studies Fischer R. In: Andra W, Nowak H, editors. MagneAsm in medicine: Berlin: Wiley-‐VCH; 1998. p. 286-‐301.
0
2,000
4,000
6,000
8,000
10,000
0 1,000 2,000 3,000 4,000 5,000 6,000 7,000 8,000 9,000
Liver: LIC < 5,000 μg/g Liver: LIC > 5,000 μg/g 2–4 fold biopsies Spleen Linear fit: LIC < 5,000 μg/g
LICblop = 1.03*LICBLS-‐33 R2 = 0.96
LIC from biomagneAc liver susceptometry (μg/gliv )
LIC from
biopsy (μg/g li
v)
LIC and cardiac iron • Poor predictor of cardiac iron and of cardiac complicaAons (arrhythmia and heart failure)
• Cardiac iron easily measured together with T2* LIC
LIC-‐ when to measure?
• Annually in TDT and transfused SCD • ?bi-‐annually in TI – no clear guidelines yet • In severe cardiac iron and other high risk paAents 3-‐6 monthly (together with cardiac T2*, LVEF, chamber sizes and mobility)
• ChelaAon is usually Atrated by ferriAn trend, however if there is no response in ferriAn there may be response in LIC in a substanAal proporAon of paAents
The cross secAonal relaAonship of serum ferriAn and LIC is less clear at high iron loads
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Better correlation between baseline serum ferritin and LIC when serum ferritin is <4000 ng/mL and when LIC is <20 mg Fe/g dw
Baseline LIC (m
g Fe/g dw)
60
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Baseline serum ferriAn (ng/mL)
0 18,000 2000 4000 6000 12,000 14,000 8000 10,000 16,000
Baseline LIC (m
g Fe/g dw)
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10
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30
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50
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Baseline serum ferriAn (ng/mL)
0 18,000 2000 4000 6000 12,000 14,000 8000 10,000 16,000
Baseline serum ferriAn category <4000 ng/mL ≥4000 ng/mL
Pearson correlaAon coefficient 0.59 0.19
Baseline LIC category <20 mg Fe/g dw ≥20 mg Fe/g dw
Pearson correlaAon coefficient 0.46 0.21
Porter et al 2014 ASH oral presentaAon 2014
Value of controlling serum ferriAn -‐ evidence in thalassaemia major
• Change in serum ferriAn over Ame
reflects change in LIC
– SequenAal evaluaAon of ferriAn
good index of chelaAon historya
• Maintenance of serum ferriAn < 2500 µg/L
– Over <me significantly correlates with
cardiac disease-‐free survivalb,c,d,e
a Olivieri NF, et al. N Engl J Med. 1994;331:574-‐578. B GabuK V and Piga A. Acta Haematol. 1996;95:26-‐36. C Telfer PT, et al. Br J Haematol. 2000;110:971-‐977. d Davis BA, et al. Blood. 2004 104: 263-‐9 e Borgna-‐PignaK et al. Haematologica; 89: 1187-‐1193
Survival Prob
ability
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FerriFn >2500 μ/L on >1/3 of occasions
FerriFn <2500 μ/L on >2/3 of occasions
Years of Follow-‐Up
.
Maintenance of Lower Ferritin Levels a Positive Indicator for Survival at UCLH (unpublished data)
NonintervenAonal LIC measurement
• SQUID= superconducAng quantum interference device
• CT – under development • MRI
– R2* i.e. 1/T2* Anderson 2000 proof of concept – R2 i.e. 1.T2 (Ferriscan) St Pierre 2005
