Histopathology 2014, 65, 561–569. DOI: 10.1111/his.12417
Iron overload secondary to cirrhosis: a mimic of hereditary haemochromatosis? Murad Abu Rajab,1 Leana Guerin,2 Pauline Lee3 & Kyle E Brown1,4,5 1
Division of Gastroenterology-Hepatology, University of Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City, IA, USA, 2Department of Pathology, University of Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City, IA, USA, 3Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA, USA, 4Program in Free Radical and Radiation Biology, University of Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City, IA, USA, and 5Iowa City Veterans Administration Medical Center, Iowa City, IA, USA Date of submission 18 December 2013 Accepted for publication 12 March 2014 Published online Article Accepted 17 March 2014
Abu Rajab M, Guerin L, Lee P & Brown K E (2014) Histopathology 65, 561–569
Iron overload secondary to cirrhosis: a mimic of hereditary haemochromatosis? Aims: Hepatic iron deposition unrelated to hereditary haemochromatosis is common in cirrhosis. The aim of this study was to determine whether hepatic haemosiderosis secondary to cirrhosis is associated with iron deposition in extrahepatic organs. Methods and results: Records of consecutive adult patients with cirrhosis who underwent autopsy were reviewed. Storage iron was assessed by histochemical staining of sections of liver, heart, pancreas and spleen. HFE genotyping was performed on subjects with significant liver, cardiac and/or pancreatic iron. The 104 individuals were predominantly male (63%), with a mean age of 55 years. About half (46%) had stainable hepatocyte iron, 2+ or less in most cases. In
six subjects, there was heavy iron deposition (4+) in hepatocytes and biliary epithelium. All six of these cases had pancreatic iron and five also had cardiac iron. None of these subjects had an explanatory HFE genotype. Conclusions: In this series, heavy hepatocyte iron deposition secondary to cirrhosis was commonly associated with pancreatic and cardiac iron. Although this phenomenon appears to be relatively uncommon, the resulting pattern of iron deposition is similar to haemochromatosis. Patients with marked hepatic haemosiderosis secondary to cirrhosis may be at risk of developing extrahepatic complications of iron overload.
Keywords: cardiomyopathy, haemosiderosis, hepcidin, HFE, pancreas
Introduction Hereditary haemochromatosis (HH) is an autosomal recessive disorder of iron metabolism. Most patients with typical HH are homozygous for the C282Y mutation of the HFE gene, with a minority of cases occurring in compound heterozygotes who carry a single copy of the C282Y mutation and a second mutation designated H63D.1 In affected individuals, excess iron accumulates in the liver, heart, pancreas Address for correspondence: K E Brown, MD, Division of Gastroenterology–Hepatology, 4553 JCP, 200 Hawkins Drive, Iowa City, IA 52242, USA. e-mail: [email protected] © 2014 John Wiley & Sons Ltd.
and other organs, where it can cause cirrhosis, cardiomyopathy and endocrinopathy. Cirrhosis itself can result in hepatic iron accumulation in the absence of an inherited defect in iron metabolism.2 The term ‘haemosiderosis’ is used to distinguish iron deposition in this context from iron overload that is the primary cause of tissue injury.3 Prior to identification of the HFE mutations, it was difficult to exclude HH definitively as a contributor in cases of cirrhosis in which excess hepatic iron was present. However, several studies have shown that iron deposition unrelated to HH is common in cirrhotic livers.4–6 In the largest series, stainable iron was present in one-third of explanted cirrhotic livers,
562
M Abu Rajab et al.
with almost 10% demonstrating hepatic iron concentrations of a magnitude thought previously to be diagnostic of HH.4 Thus, hepatic haemosiderosis in the context of cirrhosis is neither rare nor is it uniformly mild. The final common pathway leading to iron accumulation in HFE-linked HH, other forms of primary iron overload and iron overload secondary to haematological diseases such as thalassaemia involves defective regulation of the iron-regulatory hormone hepcidin.7 A small peptide produced by the liver, the expression of hepcidin is regulated by various physiological stimuli, including transferrin saturation, inflammatory cytokines and hypoxia.8 Hepcidin diminishes the uptake of dietary iron from the intestine by blocking the egress of iron from enterocytes; it also causes macrophages to retain iron derived from catabolism of senescent red blood cells. Given that dysregulation of hepcidin expression has also been implicated in the pathogenesis of iron overload secondary to cirrhosis,9–11 this suggests that iron accumulation in this condition may affect organs in addition to the liver. A small number of patients with end-stage liver disease and heavy cardiac and pancreatic iron in the absence of explanatory HFE mutations have been reported in two case series12,13 but, to date, no studies have systematically addressed the question of extrahepatic haemosiderosis in cirrhosis. The aim of this study was therefore to assess extrahepatic iron deposition in a consecutive series of patients with cirrhosis who underwent autopsy at our institution.
were not evaluated. All sections were evaluated by a single pathologist (L.G.). Hepatocellular iron deposition was graded using a standard scoring system (0–4+).14 Iron in Kupffer cells and biliary epithelium was assessed separately from hepatocyte iron and graded on a semiquantitative scale as none, minimal, mild, moderate or marked. In heart, pancreas and spleen, the percentage of cells with stainable iron and intensity of the staining were recorded. Examples of the grading of iron in Kupffer cells, biliary epithelium, heart, pancreas and spleen are provided in the Supplementary information. HFE GENOTYPING
Genotyping for the C282Y mutation was performed using fixed liver tissue as a source of genomic DNA, in cases with 3+ or greater liver iron or with significant cardiac or pancreatic iron. DNA was isolated from tissue blocks using the QIAamp DNA FFPE tissue kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. Genomic DNA (50 ng) was used to amplify the C282Y region of the HFE gene using the primers HFE 4F (50 -AGTTCCAGTCTT CCTGGCAA-30 ) and HFE 4R (50 -AGCTCCTGGCTCT CATCAGT-30 ) and buffer conditions described previously,15 for one cycle of denaturation at 95°C, and 35 cycles at 95°C for 1 min, 57°C for 30 s and 72°C for 30 s. The amplified PCR product was purified
Table 1. Characteristics of autopsied cases with cirrhosis at the University of Iowa, 2000–09
Materials and methods A review was performed of all autopsies performed at the University of Iowa during 2000–09 to identify autopsies on patients ≥18 years of age in which there was a histological diagnosis of cirrhosis. These cases comprised the study group. Demographic, clinical and pathological data were obtained from the electronic medical records of these individuals. The study was approved by the institutional review board of the University of Iowa, which waived consent for HFE genotyping on autopsy material.
IRON STAINING
Paraffin-embedded blocks of liver, heart, pancreas and spleen were sectioned and stained for storage iron using Perl’s Prussian Blue method. Blocks from the central nervous system and/or pituitary gland were unavailable in most cases, hence these tissues
Age: mean (range), years Sex (M/F)
55.1 (30–95) 65/39
Cause of underlying liver disease Chronic hepatitis C
27
Chronic hepatitis B
4
Chronic hepatitis B and C
3
Alcoholic liver disease
26
Nonalcoholic steatohepatitis
11
a1-Antitrypsin deficiency
3
Primary sclerosing cholangitis
2
Primary biliary cirrhosis
1
Autoimmune hepatitis
1
Unknown or not investigated
26
© 2014 John Wiley & Sons Ltd, Histopathology, 65, 561–569.
WM
WM
WM
WM
WM
WM
1
2
© 2014 John Wiley & Sons Ltd, Histopathology, 65, 561–569.
3
4
5
6
59
69
53
52
49
41
NASH
HCV
HCV
NASH
HCV/alcohol
a1-antitrypsin deficiency
Ethnicity/ Underlying liver Case Sex Age disease
wt/wt
C282Y/ wt
wt/wt
C282Y/ wt
H63D/ wt
H63D/ wt
70
90
90
90
90
95
HFE Pancreas genotype iron (%)
Table 2. Characteristics of cirrhotic individuals with 4+ hepatic iron
Yes
No
Yes
No
No
No
Mild fibrosis
None
None
None
None
Mild fibrosis
10
30
90
0
70
95
Cardiac Pancreatic iron Diabetes histopathology (%)
No; enlarged LV with normal wall thickness and systolic function on echo
No; normal echo
Not assessed
No; normal echo
No; normal echo
No
Cardiac dysfunction
0
2
Mild fibrosis in right ventricle
0
0
0
Iron overload secondary to cirrhosis: a mimic of hereditary haemochromatosis? Murad Abu Rajab,1 Leana Guerin,2 Pauline Lee3 & Kyle E Brown1,4,5 1
Division of Gastroenterology-Hepatology, University of Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City, IA, USA, 2Department of Pathology, University of Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City, IA, USA, 3Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA, USA, 4Program in Free Radical and Radiation Biology, University of Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City, IA, USA, and 5Iowa City Veterans Administration Medical Center, Iowa City, IA, USA Date of submission 18 December 2013 Accepted for publication 12 March 2014 Published online Article Accepted 17 March 2014
Abu Rajab M, Guerin L, Lee P & Brown K E (2014) Histopathology 65, 561–569
Iron overload secondary to cirrhosis: a mimic of hereditary haemochromatosis? Aims: Hepatic iron deposition unrelated to hereditary haemochromatosis is common in cirrhosis. The aim of this study was to determine whether hepatic haemosiderosis secondary to cirrhosis is associated with iron deposition in extrahepatic organs. Methods and results: Records of consecutive adult patients with cirrhosis who underwent autopsy were reviewed. Storage iron was assessed by histochemical staining of sections of liver, heart, pancreas and spleen. HFE genotyping was performed on subjects with significant liver, cardiac and/or pancreatic iron. The 104 individuals were predominantly male (63%), with a mean age of 55 years. About half (46%) had stainable hepatocyte iron, 2+ or less in most cases. In
six subjects, there was heavy iron deposition (4+) in hepatocytes and biliary epithelium. All six of these cases had pancreatic iron and five also had cardiac iron. None of these subjects had an explanatory HFE genotype. Conclusions: In this series, heavy hepatocyte iron deposition secondary to cirrhosis was commonly associated with pancreatic and cardiac iron. Although this phenomenon appears to be relatively uncommon, the resulting pattern of iron deposition is similar to haemochromatosis. Patients with marked hepatic haemosiderosis secondary to cirrhosis may be at risk of developing extrahepatic complications of iron overload.
Keywords: cardiomyopathy, haemosiderosis, hepcidin, HFE, pancreas
Introduction Hereditary haemochromatosis (HH) is an autosomal recessive disorder of iron metabolism. Most patients with typical HH are homozygous for the C282Y mutation of the HFE gene, with a minority of cases occurring in compound heterozygotes who carry a single copy of the C282Y mutation and a second mutation designated H63D.1 In affected individuals, excess iron accumulates in the liver, heart, pancreas Address for correspondence: K E Brown, MD, Division of Gastroenterology–Hepatology, 4553 JCP, 200 Hawkins Drive, Iowa City, IA 52242, USA. e-mail: [email protected] © 2014 John Wiley & Sons Ltd.
and other organs, where it can cause cirrhosis, cardiomyopathy and endocrinopathy. Cirrhosis itself can result in hepatic iron accumulation in the absence of an inherited defect in iron metabolism.2 The term ‘haemosiderosis’ is used to distinguish iron deposition in this context from iron overload that is the primary cause of tissue injury.3 Prior to identification of the HFE mutations, it was difficult to exclude HH definitively as a contributor in cases of cirrhosis in which excess hepatic iron was present. However, several studies have shown that iron deposition unrelated to HH is common in cirrhotic livers.4–6 In the largest series, stainable iron was present in one-third of explanted cirrhotic livers,
562
M Abu Rajab et al.
with almost 10% demonstrating hepatic iron concentrations of a magnitude thought previously to be diagnostic of HH.4 Thus, hepatic haemosiderosis in the context of cirrhosis is neither rare nor is it uniformly mild. The final common pathway leading to iron accumulation in HFE-linked HH, other forms of primary iron overload and iron overload secondary to haematological diseases such as thalassaemia involves defective regulation of the iron-regulatory hormone hepcidin.7 A small peptide produced by the liver, the expression of hepcidin is regulated by various physiological stimuli, including transferrin saturation, inflammatory cytokines and hypoxia.8 Hepcidin diminishes the uptake of dietary iron from the intestine by blocking the egress of iron from enterocytes; it also causes macrophages to retain iron derived from catabolism of senescent red blood cells. Given that dysregulation of hepcidin expression has also been implicated in the pathogenesis of iron overload secondary to cirrhosis,9–11 this suggests that iron accumulation in this condition may affect organs in addition to the liver. A small number of patients with end-stage liver disease and heavy cardiac and pancreatic iron in the absence of explanatory HFE mutations have been reported in two case series12,13 but, to date, no studies have systematically addressed the question of extrahepatic haemosiderosis in cirrhosis. The aim of this study was therefore to assess extrahepatic iron deposition in a consecutive series of patients with cirrhosis who underwent autopsy at our institution.
were not evaluated. All sections were evaluated by a single pathologist (L.G.). Hepatocellular iron deposition was graded using a standard scoring system (0–4+).14 Iron in Kupffer cells and biliary epithelium was assessed separately from hepatocyte iron and graded on a semiquantitative scale as none, minimal, mild, moderate or marked. In heart, pancreas and spleen, the percentage of cells with stainable iron and intensity of the staining were recorded. Examples of the grading of iron in Kupffer cells, biliary epithelium, heart, pancreas and spleen are provided in the Supplementary information. HFE GENOTYPING
Genotyping for the C282Y mutation was performed using fixed liver tissue as a source of genomic DNA, in cases with 3+ or greater liver iron or with significant cardiac or pancreatic iron. DNA was isolated from tissue blocks using the QIAamp DNA FFPE tissue kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. Genomic DNA (50 ng) was used to amplify the C282Y region of the HFE gene using the primers HFE 4F (50 -AGTTCCAGTCTT CCTGGCAA-30 ) and HFE 4R (50 -AGCTCCTGGCTCT CATCAGT-30 ) and buffer conditions described previously,15 for one cycle of denaturation at 95°C, and 35 cycles at 95°C for 1 min, 57°C for 30 s and 72°C for 30 s. The amplified PCR product was purified
Table 1. Characteristics of autopsied cases with cirrhosis at the University of Iowa, 2000–09
Materials and methods A review was performed of all autopsies performed at the University of Iowa during 2000–09 to identify autopsies on patients ≥18 years of age in which there was a histological diagnosis of cirrhosis. These cases comprised the study group. Demographic, clinical and pathological data were obtained from the electronic medical records of these individuals. The study was approved by the institutional review board of the University of Iowa, which waived consent for HFE genotyping on autopsy material.
IRON STAINING
Paraffin-embedded blocks of liver, heart, pancreas and spleen were sectioned and stained for storage iron using Perl’s Prussian Blue method. Blocks from the central nervous system and/or pituitary gland were unavailable in most cases, hence these tissues
Age: mean (range), years Sex (M/F)
55.1 (30–95) 65/39
Cause of underlying liver disease Chronic hepatitis C
27
Chronic hepatitis B
4
Chronic hepatitis B and C
3
Alcoholic liver disease
26
Nonalcoholic steatohepatitis
11
a1-Antitrypsin deficiency
3
Primary sclerosing cholangitis
2
Primary biliary cirrhosis
1
Autoimmune hepatitis
1
Unknown or not investigated
26
© 2014 John Wiley & Sons Ltd, Histopathology, 65, 561–569.
WM
WM
WM
WM
WM
WM
1
2
© 2014 John Wiley & Sons Ltd, Histopathology, 65, 561–569.
3
4
5
6
59
69
53
52
49
41
NASH
HCV
HCV
NASH
HCV/alcohol
a1-antitrypsin deficiency
Ethnicity/ Underlying liver Case Sex Age disease
wt/wt
C282Y/ wt
wt/wt
C282Y/ wt
H63D/ wt
H63D/ wt
70
90
90
90
90
95
HFE Pancreas genotype iron (%)
Table 2. Characteristics of cirrhotic individuals with 4+ hepatic iron
Yes
No
Yes
No
No
No
Mild fibrosis
None
None
None
None
Mild fibrosis
10
30
90
0
70
95
Cardiac Pancreatic iron Diabetes histopathology (%)
No; enlarged LV with normal wall thickness and systolic function on echo
No; normal echo
Not assessed
No; normal echo
No; normal echo
No
Cardiac dysfunction
0
2
Mild fibrosis in right ventricle
0
0
0
