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Duration of Hepatic Iron Exposure Increases the Risk of Significant Fibrosis in Hereditary Hemochromatosis: A New Role for Magnetic Resonance Imaging

The American Journal of Gastroenterology

April 2005

K. Olynyk, M.D. 1 , G. St. Pierre, Ph.D. 1 , S. Britton, Ph.D. 1 , M. Brunt, M.D. 1 , and Bruce R. Bacon, M.D. 1

1School of Medicine and Pharmacology; and School of Physics, University of Western Australia, Nedlands, Western Australia; Department of Gastroenterology, Fremantle Hospital, Western Australia; The Western Australian Institute of Medical Research, Nedlands, Western Australia; Division of Gastroenterology and Hepatology, Department of Internal Medicine; and Department of Pathology, Saint Louis University Liver Center, Saint Louis University School of Medicine, St. Louis, Missouri

ABSTRACT

OBJECTIVES: Hepatic fibrosis is a complication of hereditary hemochromatosis. The aim of this study was to determine whether the product of the magnitude and duration of hepatic iron exposure is related to the risk of significant fibrosis.

METHODS: Receiver-operating characteristic curve analysis to determine the utility of hepatic iron concentration (HIC) and age in the diagnosis of low- or high-grade fibrosis was undertaken retrospectively in 60 subjects who had undergone liver biopsy for assessment of hereditary hemochromatosis. A prospective pilot study was then conducted in 10 additional subjects to evaluate utility of magnetic resonance imaging (MRI) measurements of HIC to predict fibrosis.

RESULTS: Eighteen subjects had high-grade fibrosis while 42 subjects had low-grade fibrosis. Hepatic iron concentration alone was highly sensitive (100%) but of limited specificity (67%) in diagnosis of high-grade fibrosis. The product of [HIC x age] had a sensitivity and specificity of 100% and 86%, respectively, for diagnosis of high-grade fibrosis. Magnetic resonance imaging measurements also provided accurate assignment of subjects into fibrosis severity groups.

CONCLUSIONS: Duration of exposure to iron is important in the development of hepatic fibrosis in hereditary hemochromatosis. The product of HIC and age is highly sensitive and specific for diagnosis of high-grade fibrosis and can be obtained using MRI.

INTRODUCTION

Hereditary hemochromatosis (HH) is an inherited disorder of iron metabolism with variable phenotypic expression that in most populations of northern European descent results from a homozygous C282Y mutation in the hemochromatosis (HFE) gene (1-8). In some HH patients, progressive iron overload can result in end-organ damage such as hepatic fibrosis or cirrhosis, arthritis, diabetes mellitus, or cardiomyopathy (2, 4, 7). The development of cirrhosis in HH patients is associated with reduced survival and a 20- to 100-fold increased risk of hepatocellular carcinoma compared with the control population (5, 9). However, survival is normal in those subjects who do not have cirrhosis (9). It is for this reason that assessment of the liver for the presence of significant fibrosis or cirrhosis is still a consideration in the evaluation of subjects with HH. While subjects with normal biochemical liver function tests and serum ferritin levels less than 1,000 mug/L are unlikely to have significant fibrosis, the remainder are usually recommended to undergo liver biopsy for quantitation of hepatic iron concentration (HIC) and assessment for fibrosis or cirrhosis (10-12).

The risk of significant fibrosis or cirrhosis has been associated with the level of HIC and concomitant alcohol consumption (13-16). Bassett et al. (13) introduced the concept of a threshold for HIC above which cirrhosis was more likely. The data of Sallie et al. (17) suggest that, in addition to HIC, an age greater than 45 yr may be a risk factor for development of significant fibrosis or cirrhosis in HH. In recent times, it has become apparent that subjects may develop significant fibrosis at a much lower HIC than previously thought (2, 15, 16). Given the slowly progressive nature of iron deposition and mild submorphological inflammation that occur in HH (18), we hypothesize that it is the extent of exposure to iron as determined by time and HIC that may be most relevant to the risk of developing significant fibrosis. The aim of this study was to determine whether the product of the magnitude and duration of hepatic iron exposure is related to the risk of significant fibrosis.

AUTHOR DISCUSSION

HFE-related HH is a slowly progressive disease of iron overload, which in some individuals results in significant hepatic fibrosis or cirrhosis (4, 8). A major management issue faced by clinicians concerns the identification of those at highest risk of cirrhosis and appropriate use of invasive investigations such as liver biopsy. In the current study, we explored the relationship between HIC, age, and risk of high-grade fibrosis and demonstrated that exposure to iron, as measured by the product of HIC and age, exhibits high sensitivity and specificity for prediction of high-grade fibrosis in HH. These observations extend the previous concept that hepatic injury in HH is predominantly the result of excessive iron levels to include duration of exposure to elevated iron stores. Duration of exposure has been shown previously to be an important risk factor for development of significant fibrosis in other liver diseases such as chronic hepatitis C (23, 24). This may explain why some HH subjects with relatively lower HIC levels may still develop significant fibrosis provided they have been exposed for a sufficient duration (2, 15). This concept is also consistent with data reported recently from a long-term natural history study of the evolution of untreated HH that indicate that iron status, defined by serum ferritin level, may remain constant at levels less than 1,000 mug/L for up to 17 yr in subjects who subsequently go on to develop cirrhosis (6).

There are potential confounding factors that can influence the development of liver disease in HH. For example, it is well recognized that cofactors such as alcohol consumption and chronic viral hepatitis may accelerate liver injury in HH (11, 16). To minimize the effect of these and other potential cofactors we excluded subjects with other inherited or acquired forms of chronic liver injury. Thus, our study population is highly selected to represent the subgroup of subjects who are homozygous for the C282Y mutation and who present phenotypically with biochemical or clinical features suggestive of iron overload and who are recommended to undergo liver biopsy by their clinicians. In clinical practice, this is likely to comprise less than 10% of adult C282Y homozygotes (2, 3, 25-27).

Measurements of HIC previously were dependent on percutaneous liver biopsy to obtain liver tissue. Clearly this would not be useful in a clinical context for noninvasive fibrosis risk profiling of subjects with HH. While criteria based on blood laboratory variables, such as ferritin level, platelet count, and AST or ALT activity, exist for assigning a prebiopsy risk of advanced fibrosis in HH, subjects who do not meet these criteria still require consideration of liver biopsy (10-12, 28). Magnetic resonance imaging offers the possibility of noninvasive accurate measurement of HIC and possible assessment of fibrosis in iron overload (19). In the current study, MRI measurement of HIC and calculation of the product of HIC and age accurately assigned the majority of study subjects into the high- and low-grade fibrosis categories, paralleling the findings of the retrospective study. Thus, addition of a threshold level for hepatic iron exposure, based on MRI measurements, of 480,000 [mug/g]x yr to existing criteria as a noninvasive risk marker for significant fibrosis warrants further evaluation, as it may further reduce the requirement for liver biopsy in assessment of significant liver injury in HH.

In conclusion, the length of time to which the liver is exposed to elevated hepatic iron levels is an important factor in the development of hepatic fibrosis in HH. The product of HIC and age is a highly sensitive and specific marker for high-grade fibrosis, and noninvasive MRI measurement of HIC can be used for such an approach.

RESULTS

Retrospective Study of HIC and Age as Determinants of High-Grade Hepatic Fibrosis

HH subjects with high-grade fibrosis were older, predominantly male, and had higher serum ferritin and HIC levels (Table 1, Fig. 1A). There was substantial overlap in HIC levels between subjects with low-grade and high-grade fibrosis (Fig. 1A). ROC curve analysis demonstrated that HIC alone had a sensitivity of 100% and specificity of 67% for diagnosis of high-grade hepatic fibrosis in HH using a cutoff value of 8,268 mug/g (Fig. 1B). When age was introduced into the analysis such that the product of HIC and age was used as a measure of exposure to iron, there was a marked reduction in overlap between the fibrosis groups (Fig. 1C). ROC curve analysis demonstrated improved specificity (86%) with preservation of sensitivity at 100% for diagnosis of high-grade fibrosis in HH using a cutoff value of 479,745 [mug/g]x yr (Fig. 1D). There were 18 female HH subjects in this study, 2 of whom had high-grade fibrosis. All female subjects were correctly assigned into high-grade or low-grade fibrosis groups using the HIC x age cutoff value of 479,745 [mug/g]x yr.

Prospective Pilot Study of MRI Measurement of HIC as a Predictor of High-Grade Fibrosis

Ten consecutively diagnosed HH subjects underwent both liver biopsy and MRI measurement of HIC. The agreement between the measurement of HIC derived from MRI and that from biopsy was consistent with a previous study (19), with all data falling within the 95% limits of agreement determined previously (19). Magnetic resonance imaging measurement of HIC and calculation of [HIC x age] assigned the study subjects into the high- and low-grade fibrosis categories with a sensitivity of 100% and a specificity of 80%, paralleling the findings of the retrospective study.

Subjects

RETROSPECTIVE STUDY The study group comprised 60 well-characterized subjects consecutively diagnosed with HH at Saint Louis University or Fremantle Hospital between 1996 and 2000. The inclusion criteria were: (1) previous liver biopsy for confirmation of diagnosis, staging of fibrosis, and measurement of HIC, (2) homozygosity for C282Y mutation in the HFE gene, and (3) exclusion of other causes of liver disease (2, 11). Serological testing was used to exclude chronic viral hepatitis B or C infection, autoimmune hepatitis, alpha-1 antitrypsin deficiency, and disease. Alcohol consumption was less than 2 standard drinks per day. Nonalcoholic steatohepatitis was excluded on the basis of liver biopsy appearance and clinico-pathological correlation. All subjects provided written informed consent and the studies were approved by the Institutional Review Board of Saint Louis University and the Human Research Ethics Committee of the University of Western Australia.

PROSPECTIVE PILOT VALIDATION STUDY To determine whether the results of the retrospective study could be validated and applied to current patients using noninvasive MRI measurement of HIC, a small group of HH subjects diagnosed consecutively between 2000 and 2003 at Fremantle Hospital and who were recommended to undergo liver biopsy were subjected to a recently described MRI method for HIC measurement, in addition to biochemical measurement of HIC (19). Magnetic resonance imaging was usually performed within 2 wk of liver biopsy and prior to phlebotomy therapy in a single MRI scanner. All subjects provided written informed consent and the study was approved by the Human Research Ethics Committee of the University of Western Australia.

Liver Biopsy: Histopathology and Determination of Hepatic Iron Content

Paraffin-embedded liver tissue was sectioned for standard assessment of liver pathology using hematoxylin and eosin staining. Fibrosis was assessed using Masson's trichrome stain and graded using the following semi-quantitative method: stage 1-no fibrosis, or only slightly enlarged portal tracts; stage 2-periportal fibrosis; stage 3-portal-portal bridging fibrosis; and stage 4-cirrhosis (20). For the purposes of this study, stages 1 and 2 were combined and termed "low-grade fibrosis" while stages 3 and 4 were combined and termed "high-grade fibrosis". The biochemical measurement of HIC was performed using either the method of Torrance and Bothwell (21) or atomic absorption spectrophotometry after wet-acid digestion (22). All HIC values were expressed as mug Fe/g dry weight.

Magnetic Resonance Imaging

MRI was conducted on a 1.5 T whole-body imaging unit (Siemens MAGNETOM Vision Plus) using a recently described method (19). This method measures the mean liver proton transverse relaxation (R2), which has a high sensitivity and specificity for prediction of HIC (19). A chest coil was used for signal detection in imaging of the phantoms. Images were acquired in half Fourier with a multislice single-spin-echo (SSE) pulse sequence, with the pulse repetition time TR = 2,500 ms, spin echo times TE = 6, 9, 12, 15, and 18 ms, and slice thickness = 5 mm. All spin-echo sequences were performed using fixed gain control.

Subjects were positioned such that the liver was located central to the chest coil. Nineteen slices were collected for each subject, with the gap between slices varied from person to person to enable entire coverage of the liver. A 1 L bag of Hartmann's solution was imaged with both the phantoms and volunteers to provide an external reference signal intensity within each image for the correction of any instrument drift.

R2 values were calculated throughout the liver by curve fitting the equation for the bi-exponential decay in transverse magnetization following an SSE pulse sequence to the voxel intensity data as a function of TE with radiofrequency field intensity-weighted spin density projection. In order to localize the R2 measurement to the vicinity of the biopsy site for comparison with biochemical measurements of HIC, a lateral region of the right lobe of the liver bounded by its surface and a sagittal plane 35 mm medial to its most lateral surface point was used to calculate a mean R2 value.

Statistical Analysis

All data are presented as the mean ± SEM unless otherwise specified. Statistical comparisons between groups were made using Student's unpaired t-test. For study of the relationship between HIC and fibrosis, the following variables were evaluated: HIC, HIC x age, and fibrosis grade (low or high). Receiver-operating characteristic (ROC) curve analysis was performed on these variables for evaluation of sensitivity and specificity in the diagnosis of fibrosis grade in HH. All statistical tests were conducted using GraphPad Prism 4.00 (GraphPad Software, San Diego, CA). Statistical significance was assigned for p< 0.05.

REFERENCES

1. Feder JN, Gnirke A, W, et al. A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis. Nat Genet 1996;13: 399-08.

CrossRef Abstract MEDLINE Abstract ISI Abstract CSA Abstract

2. Olynyk JK, Cullen DJ, Aquilia S, et al. A population-based study of the clinical expression of the hemochromatosis gene. N Engl J Med 1999;341: 718-24.

CrossRef Abstract MEDLINE Abstract ISI Abstract CSA Abstract

3. Beutler E, Felitti VJ, Koziol JA, et al. Penetrance of 845G-A (C282Y) HFE hereditary haemochromatosis mutation in the USA. Lancet 2002;359: 211-18.

CrossRef Abstract MEDLINE Abstract ISI Abstract CSA Abstract

4. on SA, Bacon BR. Hereditary hemochromatosis: Update for 2003. J Hepatol 2003;38(Suppl 1):S14-23.

CrossRef Abstract

5. Elmberg M, Hultcrantz R, Ekbom A, et al. Cancer risk in patients with hereditary hemochromatosis and their first-degree relatives. Gastroenterology 2003;125: 1733-41.

MEDLINE Abstract ISI Abstract

6. Olynyk JK, Hagan SE, Cullen DJ, et al. Evolution of untreated hereditary hemochromatosis in the Busselton population: A 17-year study. Mayo Clin Proc 2004;79: 309-13.

MEDLINE Abstract ISI Abstract

7. Galhenage SP, Viiala CH, Olynyk JK. Screening for hemochromatosis: Patients with liver disease, families, and populations. Curr Gastroenterol Rep 2004;6: 44-51.

MEDLINE Abstract

8. Pietrangelo A. Hereditary hemochromatosis: A new look at an old disease. N Engl J Med 2004;350: 2383-97.

CrossRef Abstract MEDLINE Abstract ISI Abstract

9. Niederau C, Fischer R, Purschel A, et al. Long-term survival in patients with hereditary hemochromatosis. Gastroenterology 1996;110: 1107-19.

MEDLINE Abstract ISI Abstract

10. Guyader D, Jacquilinet C, Moirand R, et al. Noninvasive prediction of fibrosis in C282Y homozygous hemochromatosis. Gastroenterology 1998;115: 929-36.

MEDLINE Abstract ISI Abstract

11. Bacon BR, Olynyk JK, Brunt EM, et al. HFE genotypes in patients with hemochromatosis and in other liver diseases. Ann Int Med 1999;130: 953-62.

MEDLINE Abstract ISI Abstract CSA Abstract

12. on ED, Brandhagen DJ, Phatak PD, et al. Serum ferritin level predicts advanced hepatic fibrosis among U.S. patients with phenotypic hemochromatosis. Ann Int Med 2003;138: 627-33.

MEDLINE Abstract ISI Abstract

13. Bassett ML, Halliday JW, LW. Value of hepatic iron measurements in early hemochromatosis and determination of the critical level associated with fibrosis. Hepatology 1986;6: 24-29.

MEDLINE Abstract ISI Abstract

14. Olynyk J, Hall P, Sallie R, et al. Computerized measurement of iron in liver biopsies: Comparison with biochemical iron measurement. Hepatology 1990;12: 26-30.

MEDLINE Abstract ISI Abstract

15. P. Is there a threshold of hepatic iron concentration that leads to cirrhosis in C282Y hemochromatosis? Am J Gastroenterol 2001;96: 567-69.

Synergy Abstract MEDLINE Abstract ISI Abstract

16. Fletcher LM, Dixon JL, Purdie DM, et al. Excess alcohol greatly increases the prevalence of cirrhosis in hereditary hemochromatosis. Gastroenterology 2002;122: 281-89.

MEDLINE Abstract ISI Abstract

17. Bridle KR, Crawford DHG, Fletcher LM, et al. Evidence for a sub-morphological inflammatory process in the liver in haemochromatosis. J Hepatol 2003;38: 521-25.

CrossRef Abstract MEDLINE Abstract

18. Sallie RW, WD, Shilkin KB. Confirmation of the efficacy of hepatic tissue iron index in differentiating genetic haemochromatosis from alcoholic liver disease complicated by alcoholic haemosiderosis. Gut 1991;32: 207-10.

MEDLINE Abstract ISI Abstract

19. St Pierre TG, PR, Chua-Anusorn W, et al. Non-invasive measurement and imaging of liver iron concentrations using proton magnetic resonance. Blood 2004 (prepublished online July 15).

20. Leicester KL, Olynyk JK, Brunt EM, et al. CD14-positive hepatic monocytes/macrophages increase in hereditary hemochromatosis. Liver International (in press).

21. Torrance JD, Bothwell TH. Iron stores. Meth Hematol 1980;1: 90-115.

22. Olynyk J, P, Fudge A, et al. Fine needle aspiration liver biopsy for measurement of hepatic iron concentration. Hepatology 1992;15: 502-6.

MEDLINE Abstract ISI Abstract

23. Poynard T, Bedossa P, Opolon P. Natural history of liver fibrosis progression in patients with chronic hepatitis C. The OBSVIRC, METAVIR, CLINIVIR, and DOSVIRC groups. Lancet 1997;349: 825-32.

CrossRef Abstract MEDLINE Abstract ISI Abstract CSA Abstract

24. Di Bisceglie AM. Natural history of hepatitis C: Its impact on clinical management. Hepatology 2000;31: 1014-18.

CrossRef Abstract MEDLINE Abstract ISI Abstract

25. Asberg A, Hveem K, Thorstensen K, et al. Screening for hemochromatosis: High prevalence and low morbidity in an unselected population of 65,238 persons. Scand J Gastroenterol 2001;10: 1108-15.

MEDLINE Abstract

26. McCune CA, Al-Jader LN, May A, et al. Hereditary haemochromatosis: Only 1% of adult HFE C282Y homozygotes in South Wales have a clinical diagnosis of iron overload. Hum Genet 2002;111: 538-43.

CrossRef Abstract MEDLINE Abstract ISI Abstract CSA Abstract

27. Andersen RV, Tybjaerg-Hansen A, Appleyeard M, et al. Hemochromatosis mutations in the general population: Iron overload progression rate. Blood 2004;103: 2914-19.

CrossRef Abstract MEDLINE Abstract ISI Abstract

28. Beaton M, Guyader D, Deugnier Y, et al. Noninvasive prediction of cirrhosis in C282Y-linked hemochromatosis. Hepatology 2002;36: 673-78.

CrossRef Abstract MEDLINE Abstract ISI Abstract