Section 5 -

Pathology of Hepatic Iron Overload Kay Washington Vanderbilt University Medical Center Nashville , TN

Iron overload is a commonly encountered problem in clinical practice and may be classified as primary (genetic), acquired, or secondary to other inherited diseases, such as hereditary anemias. Dietary iron is absorbed primarily by enterocytes in the duodenum, transported out of the intestine to the portal blood stream where it is bound to apotransferrin, and transported to the liver. It is therefore not surprising that excessive deposition of iron in liver is commonly encountered in liver biopsies, and although there is broad overlap in the hepatopathology of iron overload, the pattern and degree of iron deposition may suggest a specific diagnosis. Genetic Conditions due to a Defect in a Gene Primarily Involved in Iron Homeostasis Primary Hereditary Hemochromatosis HFE-associated hereditary hemochromatosis (Type 1); autosomal recessive Non-HFE-associated hereditary hemochromatosis Juvenile hemochromatosis (Type 2); hepcidin or hemojuvelin mutations Transferrin receptor 2 mutations (Type 3) Ferroportin mutations (Type 4); autosomal dominant Acquired Conditions Chronic liver disease Chronic viral hepatitis (Hepatitis C and B) Non-alcoholic fatty liver disease Alcoholic liver disease Cirrhosis Porphyria cutanea tarda Blood Diseases Related to Iron Overload Thalassemia Hereditary spherocytosis Chronic hemolytic anemias Post-transfusional iron overload Definition of Hemochromatosis The term "hemochromatosis" was originally used to describe iron accumulation not accounted for by other diseases and leading to organ damage. However, recognition of mutations in the HFE gene as the basis for most cases of hereditary hemochromatosis (HH) has led to a broader usage to include patients with biochemical and genetic evidence for the disorder, without clinical manifestations. HH is now defined as an autosomal recessive disorder of iron metabolism due to two mutant HFE alleles. However, this definition does not account for conditions with an inherited hemochromatosis-like phenotype without mutations in the HFE gene (non-HFE hemochromatosis), which account for roughly 10% of cases. [1] Genetics of Hereditary (HFE) Hemochromatosis HH due to HFE mutations is one of the most common genetic disorders in Caucasians, with a prevalence of nearly 1 in 200. The two most common mutations are designated C282Y (85-90% of affected individuals) and H63D, and account for ~85% of autosomal recessive HH. The C282Y mutation prevents formation of a disulfide bond essential for binding of the protein to β2-microglobulin and thus prevents transport of the HFE protein to the cell surface. The H63D mutation affects binding of the HFE to the transferrin receptor and results in a less severe phenotype; homozygosity for H63D mutation accounts for 2% or less of HH [2]. From 8 to 18% of northern, central, and western Europeans are heterozygotes for the C282Y mutation. The high frequency of this mutation and data from population studies suggests origin from a common ancestor in northwest Europe before 4000 BC [3]. It is postulated that heterozygotes had a selective advantage because increased dietary iron absorption was protective against iron deficiency. Selection against homozygotes is minimal, because overt disease manifestations only develop after the reproductive period is over [2]. Ferroportin disease is inherited in an autosomal dominant fashion [4]. TfR2-associated hemochromatosis and most cases of juvenile hemochromatosis are inherited as autosomal recessive disorders [1]. Clinical Features Many patients with HFE mutations are now identified at an asymptomatic stage when hyperferritinemia is discovered upon routine testing. Individuals with HFE HH are asymptomatic until significant accumulation of iron occurs in liver and other organs, a process which takes decades; men typically present in their 40s or 50s, with women presenting a decade later because of the protective effects of menstruation. Skin pigmentation is less frequently found with earlier diagnosis than in the past. Weakness, lethargy, abdominal pain, and arthropathy are common non-specific presenting symptoms [5]. In late stages, iron deposition in pancreatic islets results in diabetes mellitus. Hypogonadism is very common. In young subjects, cardiac manifestations may be the presenting feature, generally as a congestive cardiomyopathy, and may cause death within a year of presentation if iron reduction is not instituted. Treatment is generally phlebotomy to reduce iron stores. Histopathology of the Liver in Iron Overload Disorders In HH due to HFE mutations, iron is preferentially deposited in hepatocytes as granular, refractile golden-brown hemosiderin. In early stages, this iron deposition is more prominent in zone 1, but as the disease progresses, involves zone 2 and zone 3 hepatocytes. Inflammation is not a feature of hemochromatosis, and fatty change is not specifically associated with the disorder. Iron also accumulates in biliary epithelial cells. The accumulation of iron causes necrosis of individual hepatocytes, releasing iron which is taken up by Kupffer cells. In contrast to HFE HH, iron deposition in ferroportin disease and hematologic disorders occurs preferentially in Kupffer cells, with involvement of hepatocytes only when present in large quantities. Fibrosis in HH occurs roughly when a threshold of ~15,000 micrograms iron/gram dry weight liver is reached. It begins in periportal areas where iron deposition is greatest. Early cirrhosis is typically micronodular and the liver is rust-colored. Risk for hepatocellular carcinoma may be up to 200 times that of the general population, and it is postulated that the presence of increased iron produces oxidation stress that leads to p53 mutations. Of note, primary liver tumors arising in HH often displays mixed hepatocellular/cholangiocarcinoma differentiation [6]. Dysplastic nodules may contain relatively little iron (iron free foci) compared to the rest of the liver. Quantification of hepatic iron by histopathology is rarely indicated, but the following semi-quantitative grading scheme [7] is helpful in surgical pathology practice: 1+ Fine granules in 5 to 10% of hepatocytes 2+ Fine granules in 10 to 50% of hepatocytes 3+ Fine and large granules in over 50% of cells 4+ Fine and coarse granules in nearly all hepatocytes Ancillary Studies Quantitative analysis of iron in liver has long been the gold standard for diagnosis of HH but is supplanted in some settings by genetic testing. HLA typing has been used as a surrogate test for HH within kindreds because of the tight linkage between the HFE gene and the HLA complex, but has largely been supplanted by testing for C282Y mutations. Quantitative iron determination is used to determine the hepatic iron index by dividing the weight of iron (micromoles per gram) in the biopsy by the patient's age in years. A level of >1.9 reliably distinguishes homozygosity for HH from the heterozygous state and alcoholic liver disease. However, the hepatic iron index may exceed 1.9 in chronic hemolytic anemia, and levels < 1.9 are seen in up to 15% of HH patients; in particular, its utility has not been established in the pediatric population. The test may be performed on formalin-fixed, paraffin-embedded tissue or fresh tissue. Differential Diagnosis The differential diagnosis of HH primarily includes chronic liver diseases associated with hepatic iron deposition, and hematologic disorders. Although iron overload from transfusions and/or chronic hemolytic anemias is usually easily distinguished from HH by the preferential deposition of iron in Kupffer cells rather than hepatocytes, a note of caution must be sounded here because of the recent descriptions of the hepatopathology of ferroportin disease. Clinical history should aid in making these distinctions, but in ambiguous cases, genetic testing may be indicated. Hepatic iron overload is commonly seen in alcoholic liver disease and cirrhosis of any type [8] and may overlap morphologically with HH. The pathogenesis of iron deposition in alcoholics is largely unknown, but increased alcohol consumption may increase intestinal iron absorption in some patients; heterozygosity for C282Y does not appear to influence hepatic iron levels or the risk of fibrosis in these patients [9]. Quantitative iron determination is usually helpful in distinguishing these entities. Heavy alcohol consumption appears to accentuate the clinical expression of hemochromatosis, greatly increasing the prevalence of cirrhosis [10]. Hepatic iron deposition in chronic viral hepatitis is typically modest, and is found primarily in Kupffer cells; inconspicuous deposits in endothelial cells may also be noted. The contribution of HFE heterozygosity and increased hepatic iron to non-alcoholic fatty liver disease is controversial, with some but not all investigators reporting increased prevalence of HFE mutations in NASH [11, 12]. Conversely, many patients with HH have histologic features of NASH [13]. It should be noted that HH by itself does not produce an inflammatory pattern of injury in the liver, and if present, this should prompt consideration of superimposed disease processes or alternative diagnoses. Primary Iron Overload Disorders [1, 2] Disorder Gene Protein Function Pathogenesis Clinical onset Population Liver Histopathology Main clinical manifestations Hereditary hemochromatosis (Type 1) HFE Regulation of iron transport in intestinal crypt cells Excessive iron uptake from intestine 40s & 50s Caucasians, mainly northern European Preferential deposition of iron in hepatocytes (periportal in early stages); spillover to Kupffer cells; fibrosis progressing to cirrhosis Liver disease; diabetes mellitus; cardiac disease; arthropathy; endocrine disease Ferroportin disease (Type 4) SLC40A1 (solute carrier family 40) Iron export from cells (macrophages, intestine, placenta) Iron retention in macrophages 40s & 50s Worldwide; many ethnic groups Preferential deposition of iron in Kupffer cells, with late spillover to hepatocytes; sinusoidal and periportal fibrosis Liver disease; mild anemia TfR2-associated hemochromatosis (Type 3) TfR2 Uptake of iron-bound transferrin; may regulate hepcidin-HFE2 activity Unclear 40s & 50s Rare; Italian, French, Portuguese, Japanese pedigrees Hepatocellular (periportal) iron accumulation; periportal fibrosis Liver disease; mild anemia; hypogonadism Hepcidin-associated hemochromatosis and juvenile iron overload syndrome (Type 2) HAMP (hepcidin antimicrobial peptide); hemojuvelin HJV/1p21 Down-regulation of iron efflux from macrophages, intestine, placenta Excess iron release from macrophages results in influx into hepatocytes 10s & 20s Diverse ethnic groups, mostly European; Greek, Italian, French, French-Canadian pedigrees Iron overload in hepatocytes, leading to fibrosis Endocrine and cardiac disease; liver disease is not a prominent feature References Pietrangelo A. Non-HFE hemochromatosis. Seminars in Liver Disease 25:450-60, 2005. Beutler E, Bothwell TH, Charlton RW, Motulsky AG. Hereditary hemochromatosis. In Scriver, CR (ed). The Metabolic and Molecular Bases of Inherited Disease, eighth edition, pp 3127-3161. New York: McGraw-Hill, 2001. Distante S, Robson KJH, Graham-Campbell J, et al. The origin and spread of the HFE-C282Y haemochromatosis mutation. Hum Genet 115:269-79, 2004. Montosi G, Donovan A, Totaro A, Garuti C, et al. Autosomal-dominant hemochromatosis is association with a mutation in the ferroportin (SLC11A3) gene. J Clin Invest 108:619-623, 2001. O'Neil J, Powell L. Clinical aspects of hemochromatosis. Seminars in Liver Disease 25:381-91, 2005. Morcos M, Dubois S, Bralet MP, Belghiti J, Degott C, Terris B. Primary liver carcinoma in genetic hemochromatosis reveals a broad histologic spectrum. American Journal of Clinical Pathology 116(5):738-43, 2001. Conn HO. Portocaval anastomosis and hepatic hemosiderin deposition: a prospective, controlled investigation. Gastroenterology 62:61-72, 1972. Ludwig JL, Hashimoto E, Porayko MK, Moyer TP, Baldus WP. Hemosiderosis in cirrhosis: a study of 447 native livers. Gastroenterology 112:882-888, 1997. Grove J, Daly AK, Burt AD, Guzail M, James OF, Bassendine MF, et al. Heterozygotes for HFE mutations have no increased risk of advanced alcoholic liver disease. Gut 43:262-266, 1998. Fletcher LM, Dixon JL, Purdie DM, Powell LW, Crawford DHG. Excess alcohol greatly increases the prevalence of cirrhosis in hereditary hemochromatosis. Gastroenterology 122:281-289, 2002. Bonkovsky HL, Jawaid Q, Tortorelli K, LeClair P, et al. Non-alcoholic steatohepatitis and iron: increased prevalence of mutations of the HFE gene in non-alcoholic steatohepatitis. J Hepatol 31:421-429, 1999. Bugianesi E, Manzini P, D'Antico S, Vanni E, et al. Relative contribution of iron burden, HFE mutations, and insulin resistance to fibrosis in nonalcoholic fatty liver. Hepatology 39:179-184, 2004. Nash S, Marconi S, Sikorska K, Naeem R, Nash G. Role of liver biopsy in the diagnosis of hepatic iron overload in the era of genetic testing. Am J Clin Pathol 118: 73-81, 2002.