Survival After Liver Transplantation in Patients With Hepatic Iron Overload: The National Hemochromatosis Transplant Registry

GASTROENTEROLOGY 2005;129:494 –503

Survival After Liver Transplantation in Patients With Hepatic Iron Overload: The National Hemochromatosis Transplant Registry KRIS V. KOWDLEY,* DAVID J. BRANDHAGEN,‡ ROBERT G. GISH,§ NATHAN M. BASS,¶ JEFFREY WEINSTEIN,储 MICHAEL L. SCHILSKY,# ROBERT J. FONTANA,** TIMOTHY MCCASHLAND,‡‡ SCOTT J. COTLER,§§ BRUCE R. BACON,¶¶ EMMET B. KEEFFE,储 储 FREDRIC GORDON,*** NAYAK POLISSAR,‡‡‡ and the National Hemochromatosis Transplant Registry *University of Washington, Seattle, Washington; ‡Mayo Clinic College of Medicine, Rochester, Minnesota; §California Pacific Medical Center, San Francisco, California; ¶University of California at San Francisco, San Francisco, California; 储Baylor University Medical Center, Dallas, Texas; #New York Weill Cornell Medical Center and Mount Sinai School of Medicine, New York, New York; **University of Michigan, Ann Arbor, Michigan; ‡‡University of Nebraska, Omaha, Nebraska; §§Rush University, Chicago, Illinois; ¶¶St. Louis University, St. Louis, Missouri; 储 Stanford University, Stanford, California; ***Lahey Clinic, Burlington, Massachusetts; ‡‡‡The Mountain-Whisper-Light Statistical Consulting, Seattle, Washington; and the NHTR Study Group

Background & Aims: Previous uncontrolled studies have suggested that patients with hepatic iron overload have a poor outcome after liver transplantation. We examined the effect of HFE mutations on posttransplantation survival in patients with hepatic iron overload. Methods: Two hundred sixty patients with end-stage liver disease and hepatic iron overload were enrolled from 12 liver transplantation centers. Hepatic iron concentration (HIC), hepatic iron index (HII), HFE mutation status, and survival after liver transplantation were recorded. Results: HFE-associated hemochromatosis (HH) defined as homozygosity for the C282Y (n ⴝ 14, 7.2%) mutation or compound heterozygosity for the C282Y/H63D (n ⴝ 11, 5.6%) mutation was identified in 12.8% of patients. Survival postliver transplantation was significantly lower among patients with HH (1-, 3-, and 5-year survival rates of 64%, 48%, 34%, respectively) compared with simple heterozygotes (C282Y/wt or H63D/wt) or wildtype patients. Patients with HH had a hazard ratio for death of 2.6 (P ⴝ .002) after adjustment for age, United Network for Organ Sharing status, year of transplantation, and either elevated HII or HIC. Non-HH patients with hepatic iron overload also had significantly decreased survival when compared with the overall population undergoing liver transplantation (OR ⴝ 1.4, 95% CI: 1.15–1.61, P < .001). Conclusions: One- and 5-year survivals after liver transplantation are significantly lower among patients with HFE-associated HH. Our data also suggest that hepatic iron overload may be associated with decreased survival after liver transplantation, even in patients without HH. Early diagnosis of hepatic iron overload using HFE gene testing and iron depletion prior to liver transplantation may improve posttrans-

plantation survival, particularly among patients with HH.

ereditary hemochromatosis (HH) is characterized by iron overload in multiple organs, in particular the liver, heart, pancreas, joints, and endocrine glands.1 Most cases of HH are associated with mutations in the HFE gene, which encodes a nonclassical HLA class I protein. Two mutations account for the overwhelming majority (85%) of cases of phenotypic HH among American patients: the homozygous C282Y mutation (C282Y⫹/⫹) or the compound heterozygous C282Y/ H63D mutation (C282Y/H63D).2 Patients with cirrhosis because of HH have an increased risk of hepatocellular carcinoma and decompensated liver disease.1,3 Survival among HH patients with cirrhosis is significantly decreased, even with iron depletion therapy via phlebotomy, primarily because of an increased risk of hepatocellular carcinoma.4 – 6 Liver transplantation has been used to treat end-stage liver disease since the initial report from Pittsburgh of 6 patients with hemochromatosis. All patients had undergone iron depletion prior to transplantation, and all patients had survived 6 months or more after liver transplantation.7 A subsequent report from a large health care financing administration database, however, suggested that hemochromatosis was associated with a significantly

H

Abbreviations used in this paper: HIC, hepatic iron concentration; HII, hepatic iron index; HH, hereditary hemochromatosis. © 2005 by the American Gastroenterological Association 0016-5085/05/$30.00 doi:10.1053/j.gastro.2005.05.004

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lower survival after liver transplantation.8 One- and 5-year survivals among patients with a pretransplantation diagnosis of “hemochromatosis” were 54% and 43%, respectively, which was lower than for patients with alcoholic or viral cirrhosis. A major limitation of the study was the lack of a definitive diagnosis of HLAlinked hemochromatosis in this cohort, possibly because there is only 1 ICD-9 code available to describe all causes of iron overload, regardless of etiology. This study included pediatric patients and patients with secondary iron overload in the hemochromatosis group. A subsequent study enrolled 37 patients from 5 liver transplantation centers with a presumed diagnosis of hereditary hemochromatosis.3 The diagnosis was restricted to patients with unexplained hepatic iron overload in a pattern consistent with hemochromatosis or a known pretransplantation diagnosis of hemochromatosis based on hepatic iron concentration (HIC) ⬎4000 ␮g/g dry weight (approximately 70 ␮mol/g) or hepatic iron index (HII) (HII ⫽ HIC [␮mol/g]/age (years)) ⬎1.9. One- and 5-year survivals in this cohort3 were disappointingly similar to that reported by Kilpe et al.8 This cohort had a higher than expected prevalence of HCC; however, infections and cardiac complications accounted for most deaths.3,9 These early studies examining outcomes after liver transplantation among patients with hepatic iron overload presumed secondary to hemochromatosis were completed prior to the era of HFE gene testing. Subsequent studies, albeit with small sample sizes, have demonstrated that most patients with hepatic iron overload in the setting of end-stage cirrhosis do not appear to have HLA-linked or classic hereditary hemochromatosis because the majority of such patients lack the characteristic C282Y homozygous or C282Y/H63D compound heterozygous genotype.10 –12 Hepatic iron overload in the setting of end-stage liver disease appears most common among patients with chronic hepatitis C and alcoholic liver disease but has also been described among patients with other types of end-stage liver disease.13,14 Some authors have suggested that hepatic iron overload is associated with a poor outcome after liver transplantation regardless of the presence or absence of HFE mutations.12,14 Based on these data, some liver transplantation programs have been excluding such patients from liver transplantation, particularly if they have evidence of cardiac iron overload.15 However, others have argued that hepatic iron overload per se is not associated with a poor outcome after liver transplantation in patients without hemochromatosis.12 A recent study of 22 subjects with suspected HH defined as homozygosity for the C282Y mutation (n ⫽ 17) or hepatic iron overload

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associated with either the HLA A3B7 haplotype or a family history of iron overload showed relatively poor outcome after liver transplantation with 1-, 3-, and 5-year survivals of 72%, 62%, and 55%, respectively.16 Increased risk of posttransplantation mortality appeared to be related to recurrent HCC in many subjects. However, this study did not control for other risk factors for decreased survival after transplantation such as United Network for Organ Sharing (UNOS) status, age, year of transplantation, or severity of hepatic iron overload. The primary goals of the current study were (1) to examine the prevalence of HFE mutations in a large multicenter cohort of United States liver transplant recipients with known or suspected hepatic iron overload and (2) to examine the relationship between HFE genotype and survival after liver transplantation. Secondary goals were to examine the causes of death after liver transplantation among patients with hepatic iron overload and to examine the relationship between HIC, HII, and survival in this cohort.

Patients and Methods Patients Twelve liver transplantation centers participated in this study. The complete list of centers and participating investigators is shown in Appendix 1. Patients who had undergone their first liver transplantation prior to 1996 were eligible to enter this study. To be included, patients were required to have one of the following: a known diagnosis of hemochromatosis or hepatic iron overload prior to liver transplantation based on one of the following criteria: compatible hepatic iron stain (as ⱖ2⫹ diffuse, multifocal, iron staining in hepatocytes with or without stainable iron in reticuloendothelial cells); HIC ⱖ4000 ␮g/g dry weight (approximately 70 ␮mol/g) or HII ⱖ1.9; ⱖ4 grams of iron removed by quantitative phlebotomy or previously unsuspected hepatic iron overload (as defined above) found in the liver explant in the absence of a known cause of iron overload, such as blood transfusion, iron-loading anemia, homozygous ␤-thalassemia, or iatrogenic iron supplementation (in the absence of iron deficiency). The subjects in the study were ascertained as follows: known or suspected diagnosis of hemochromatosis prior to transplantation based on HII or HIC (n ⫽ 46, 17.7%) or iron overload discovered at liver transplantation without previous suspicion of iron overload (n ⫽ 214, 82.3%). Because the inclusive time period of the study was prior to the identification of the HFE gene, HFE mutation status was unknown in the study subjects at the time of transplantation. Paraffin blocks from liver explants were sent to the University of Washington, and HIC and HII measurement and HFE genotyping were performed using previously described techniques.17,18 Data were collected at the participating centers using a custom-

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ized Microsoft Access database (Microsoft Corporation, Redmond, WA) and sent electronically to the central database at the University of Washington. The following data were collected on all subjects: age at the time of transplantation, sex, UNOS status (using the old system of 1, 2a, 2b, and 3), HFE genotype, date of last follow-up, year of transplantation, and transplantation center. The following key data were collected wherever possible (given that this was a retrospective study, data were not available in all subjects): HIC and HII, presence or absence of hepatocellular carcinoma, phlebotomy therapy pretransplantation, pretransplantation diagnosis of iron overload, and cause of death. Exclusion criteria included the following: retransplantation, inability to perform HFE genotyping (lack of tissue or inability to obtain DNA from paraffin tissue [⬍5% of subjects]), or incomplete key data provided by the participating center. Inability to perform HFE genotyping most commonly occurred because subjects were deceased and because the retrospective nature of the study precluded HFE mutation analysis in some cases. The study was approved by the institutional review boards of all participating centers that enrolled patients into the study.

Statistical Methods Cumulative survival estimates and survival curves for the entire group and subgroups were calculated using the Kaplan–Meier method.19 The curves were compared between groups using the log-rank test. The 1, 3, and 5year survivals in the groups were tabulated along with their standard errors based on Greenwood’s formula.20 We compared mean HII and HIC among the genetic groups using normal-based ANOVA. Before carrying out the ANOVA, HII and HIC were transformed as log (1 ⫹ HII) and log (30 ⫹ HIC), respectively, to improve normality of distribution. The proportion of cancer cases in the genetic groups was compared using Fisher exact test. We developed a multivariate model for survival using the Cox proportional hazards method. Because the Kaplan– Meier survival curves for the UNOS groups suggested nonproportionality of hazards over time, we stratified on the UNOS categories in the Cox models rather than including UNOS as a covariate. We also compared the survival among the non-HH patients in this study (defined below as groups 3 ⫹ 4 ⫹ 5, n ⫽ 235) to survival among all adult patients undergoing a first liver transplantation at the 12 participating transplantation centers between 1990 and 1996 (n ⫽ 5493) to examine whether survival in the non-HH patients with hepatic iron overload was lower than the remaining patients undergoing a first liver transplantation. The statistical significance of the differences in survival rates between groups 3 ⫹ 4 ⫹ 5 (n ⫽ 235) and the remaining patients (n ⫽ 5493) at each time point (1, 3, 5 years) is based on the ␹2 test, using the baseline cohort sample sizes and the estimated Kaplan–Meier survival proportions in the 2 samples. Odds ratios and confidence intervals are based on binomial

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Table 1. Characteristics of the Study Population Variable

Number

Sex F M Age at transplantation Age group 0–39 years 40–49 years 50–59 years 60⫹ years Mean HII (␮mol/g/age[y]) HII ⱕ1.9 HII ⬎1.9 Mean HIC (␮mol/g dry weight) HIC ⱕ70 ␮mol/g HIC ⬎70 ␮mol/g Status Alive at last follow-up Deceased Years post transplantation UNOS 1 2a 2 or 2b 3 or 4 HFE mutation status C282Y homozygote C282Y/H63D CPD Het C282Y heterozygote H63D heterozygote H63D homozygotes Wild type

254

259 259

197

196

Mean N

SD or %

52 202 50.6

20% 80% 10.2

34 85 98 42 2.3 97 100 117 70 126

13% 33% 39% 16% 2.1 49% 51% 111 36% 64%

260

260 232

147 113 3.9

56.5% 43.5% 3.2

23 37 79 93

10% 16% 34% 40%

14 11 23 36 4 106

7% 6% 12% 19% 2% 54%

195

NOTE. Total N ⫽ 260.

proportions. This approach was chosen because of lack of information on standard errors of survival proportions in the cohort. The P values and confidence intervals are apt to be slightly anticonservative because of censoring in both groups.

Results The characteristics of the study population are shown in Table 1. The mean age of the patients was 51 years, and 80% were male. HFE genotyping was available in 195 of the 260 patients (75%). The prevalence of HFE mutations in those with genotype available was 46%. Twenty-five patients (13% of those genotyped) had HH, as defined by presence of homozygous C282Y (n ⫽ 14) or compound heterozygous C282Y/H63D mutations (n ⫽ 11). Fifty-nine (30%) patients were “simple” heterozygotes, and 106 were wild type for HFE (n ⫽ 106, 54%). Among HFE heterozygotes, 23 (12%) were C282Y heterozygotes, and 36 (18%) were H63D heterozygotes; 4 (2%) were H63D homozygotes. Among patients with available data on pretransplantation diagnoses, 46 patients (18%) were classified as having hemochromatosis, 32 (12%) as having hepatitis C, and 18

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Table 2. Relationship Between HFE Mutations Status and Post Transplant Survival 1 year (%)

3 years (%)

5 years (%)

HFE status

N

%

Survival

SE

Survival

SE

Survival

SE

Group 1: C282Y homozygotea Group 2: Compound heta Group 3: Other HFEb Group 4: Wild typea Group 5: Missinga Group 1 ⫹ 2c Group 3 ⫹ 4c Comparison to Group 1 ⫹ 2:d Group 3 ⫹ 4 ⫹ 5d Group 3a: C282Y hete Group 3b: H63D hete Group 3c: H63D homozygotee

14 11 63 107 65 25 170

5 4 24 41 25 10 65

64 64 84 78 81 64 80

13 15 5 4 5 10 3

43 55 74 69 71 48 71

13 15 6 5 6 10 4

32 33 61 65 61 34 64

14 15 7 5 7 10 4

235 23 36 4

90 9 14 2

81 87 81 100

3 7 7 0

71 77 72 75

3 9 8 22

63 66 56 75

3 11 9 22

NOTE. Significant differences occur because of the contrast in survival between groups 1 and 2 vs 3, 4, and 5. *P value based on the log-rank test. The null hypothesis is that all of the HFE groups compared have an identical survival rate over time. Survival rates at 1, 3, and 5 years are presented as illustrative, but P values are based on the continuous survival experience throughout the entire observation period. aP ⫽ .02. bC282/wt, H63D/wt, H63D/H63D. cP ⫽ .0007. dP ⫽ 0008. eP ⫽ .8.

(7%) as having alcoholic liver disease; the etiology of liver disease was unspecified or listed as cryptogenic in 60 patients (23%). Only 20 of 46 patients diagnosed as having hemochromatosis prior to liver transplantation (43%) were confirmed to have HH based on HFE genotyping. Data on presence or absence of liver cancer were present in 252 of 260 patients (97%). Liver cancer was present in 29 of 252 patients (11.5%). There was a higher prevalence of liver cancer among C282Y homozygotes (29%) compared with simple heterozygotes (13%) or wild-type patients (10%), although the difference was not statistically significant (P ⫽ .2). Data on HIC or HII were available in 197 of 260 (76%) patients. Multiple measurements of HIC were available in 11%; the mean weight per sample was 2.77 mg (92% of samples were ⬎1-mg dry weight. HIC was ⬎70 ␮mol/g dry weight in 126 of 197 (64%) patients; HII was ⬎1.9 in 100 of 197 (51%) patients. Relationship Between HFE Genotype and Survival The actual 1-year survival by HFE genotype is shown in Table 2. Cumulative survival differed significantly among genotypes (P ⫽ .02) with 1-, 3-, and 5-year survivals among C282Y homozygotes (64%, 43%, 32%, respectively) and C282Y/H63D compound heterozygotes (64%, 55%, 33%, respectively) lower than among HFE heterozygotes (84%, 74%, 61%, respectively) and wild-type patients (78%, 69%, 65%, respectively). There was no significant difference in cumulative

survival between C282Y heterozygotes and C282Y/ H63D compound heterozygotes (P ⫽ .99). There was also no significant difference in cumulative survival between C282Y heterozygotes (n ⫽ 23; 1-, 3-, and 5-year rates of 87%, 77%, 66%, respectively), H63D heterozygotes (n ⫽ 36; 81%, 72%, 56%, respectively) and H63D homozygotes (n ⫽ 4; 100%, 75%, 75%, respectively; P ⫽ .8). Therefore, the C282Y homozygous and C282Y/ H63D compound heterozygous genotypes were combined in the survival analysis as were the HFE heterozygous and wild-type groups. The overall cumulative survival for the entire cohort is shown in Figure 1. The survival curves for the HH, simple HFE heterozygote, and wild-type groups are shown in Figure 2. The combined survival curve for the C282 homozygote and C282Y/H63D compound heterozygote group compared with simple HFE heterozygote and wild-type groups is shown in Figure 3. Patients in whom HFE genotype was unavailable (classified as missing in Table 2) had 1-, 3-, and 5-year survivals that were comparable with simple heterozygotes and wild-type patients. We also compared the estimated 1-, 3-, and 5-year survivals between the non-HH patients in this study (groups 3 ⫹ 4 ⫹ 5 [n ⫽ 235]) and the remaining patients undergoing a first liver transplantation at the participating 12 centers during the time period of the study (n ⫽ 5493). As shown in Table 3, non-HH patients with hepatic iron overload undergoing liver transplantation appear to have significantly decreased 5-year

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Figure 1. Kaplan–Meier estimates for survival after liver transplantation in the overall cohort of patients with hepatic iron overload (n ⫽ 260).

survival after liver transplantation (63% vs 72%, respectively P ⫽ .003, OR ⫽ 1.51) compared with the remaining patients. Relationship Between HIC, HII, and Survival The effect of hepatic iron content on survival after liver transplantation was also examined in detail. HIC and HII were not significantly different among C282Y homozygotes and C282Y/H63D compound heterozygotes compared with patients in other genotype groups, as shown in Table 4. There was no significant difference in survival between patients with HIC ⬎70-␮mol/g dry weight compared with those with HIC ⱕ70-␮mol/g dry

Figure 2. Kaplan–Meier estimates for survival after liver transplantation among patients based on HFE genotype. C282Y ⫹/⫹, C282Y homozygous; CPD Het, C282Y/H63D compound heterozygote; Simple het, C282Y/wt or H63D/wt; Wt: wt/wt.

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weight or between patients with HII ⬎1.9 compared with those with HII ⱕ1.9, although overall survival was marginally better among patients with HIC ⱕ70 ␮mol/g and HII ⱕ1.9 (Figure 4A and 4B). HIC was also not significantly associated with survival when examined as a continuous variable (data not shown). Iron depletion therapy prior to liver transplantation was performed in only 12 of 260 patients (4.6%). Furthermore, only 16% of patients with HH had iron depletion prior to liver transplantation. There were similar posttransplantation survival rates between patients who had undergone iron depletion prior to transplantation compared with the rest of the study population (P ⫽ .8), although the proportion of patients treated with iron depletion was too small to support a definitive statement. A multivariate Cox proportional hazards regression model was fitted to the data to identify factors independently associated with posttransplantation survival. The model included genetic group (HH vs non-HH), UNOS status (as a stratifying factor), and year of transplantation (before 1991 vs after 1991), the three variables significantly associated with survival in univariate analysis (log-rank test). In addition, the model included age (years) and either HII or HIC (each in binary form). Based on this model, patients with HH had a hazard ratio for death of 2.6 (95% CI: 1.4 – 4.7, P ⫽ .002). A specific cause of death was identified in 83 of 115 (72%) patients who died during posttransplantation fol-

Figure 3. Kaplan–Meier estimates for survival after liver transplantation among patients with HH (defined as C282Y homozygous or C282Y/H63D compound heterozygous [group 1 and group 2]) compared with HFE heterozygotes (C282Y/wt or H63D wt) and patients wild type for HFE [group 3 and group 4].

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Table 3. Comparison of the Survival Among non-HH Patients With Iron Overload to Survival of Other Patients at the 12 Transplant Centers During the Period of 1990 –1996 1 year (%)

Groups 3 ⫹ 4 ⫹ 5 Whole population minus 260 patients in the NHTR study OR (95% CI)a P valueb aOdds

3 years (%)

5 years (%)

N

Survival

SE

Survival

SE

Survival

SE

235 5493

81 84

3

71 76

3

63 72

3

1.25 (0.90–1.75) .2

1.30 (0.97–1.73) .08

1.51 (1.56–1.99) .003

ratio for death for the 3 ⫹ 4 ⫹ 5 group relative to the group of the whole population minus 260 in the study. the null-hypothesis that the survival rates are the same in both groups.

bTesting

low-up. The causes of death in the study cohort are listed in Table 5 and separately for HH and non-HH patients in Table 6. There was no significant difference in the distribution of cause of death (P ⫽ .19). Among patients with an identified cause of death, posttransplantation mortality was primarily related to infection and cardiac causes, which accounted for 52% of posttransplantation mortality; infections were described as bacterial in 5.5%, fungal in 6.4%, viral in 4.6%, and unspecified in 12.7%, respectively. Cardiac complications were listed as a cause of death in 15% of patients.

Discussion The current report, to our knowledge, is the largest study to date examining survival after liver transplantation in a multicenter, nationwide cohort of patients with end-stage liver disease and hepatic iron overload. Moreover, this is the first detailed study examining the relationship between HFE mutations and outcome

after liver transplantation while controlling for UNOS status and other prognostic factors. Our study demonstrated unequivocally that patients in the United States with HFE-associated hemochromatosis, defined by the presence of the homozygous C282Y mutation or the C282Y/H63D compound heterozygous mutation, have a disappointing outcome after liver transplantation, prior to 1996 when compared with patients with hepatic iron overload and other HFE mutation patterns, even after adjustment for confounding factors such as UNOS status and year of transplantation. The majority of patients with HFE-associated hemochromatosis were not identified prior to liver transplantation, despite the expertise in liver disease among the study sites and the investigators, underscoring the difficulty in establishing the diagnosis of this condition in the setting of advanced liver disease. The infrequency of the diagnosis of hemochromatosis prior to liver transplantation is likely due in part to the lack of specificity of serum iron studies to identify pa-

Table 4. Hepatic Iron Concentration and Hepatic Iron Index Among Different HFE Genotype Groups (for Patients With Available HFE Genotype) HII (HIC/age[y]) HFE status

N

Mean

SD P⫽

Group Group Group Group

1: 2: 3: 4:

C282Y homozygote Compound het Other HFEb Wild type

13 10 57 98c

HIC (␮mol/g dry wt) Median

Mean

3.4 2.3 2.4 2.4

Group 3a: C282Y het Group 3b: H63D het Group 3c: H63D homozygote

23 155

2.9 2.4

3.3 2.6 2.1 1.9

196 109 117 119

168 61 104 114 P ⫽ .4a

212 131 97 93

2.4 2.2

2.8 2.0

158 138

118 110 P ⫽ .3a

132 95

3.2 1.3 0.7

2.2 2.1 1.3

157 96 70

P ⫽ .3a 21 33 3

3.2 2.0 1.5

Median .7a

2.9 1.2 2.2 2.1 P ⫽ .5a

Group 1 ⫹ 2 Group 3 ⫹ 4

SD P⫽

.9a

NOTE. Data for patients with available HFE genotype. aP value based on an F test comparing log(1⫹HII) or log(30⫹HIC) between HFE groups. b(C282/wt, H63D/wt, H63D/H63D). c97 observations available for HIC (age missing for one of the subjects).

148 58 40

106 97 65

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Table 5. Cause of Death After Liver Transplantation Among Patients With Hepatic Iron Overload Cardiac Infection Cardiac and Infection Malignancy (All types) Recurrent HCC Other

17 19 7 11 5 24

20% 23% 8% 13% 6% 29%

with hepatic iron overload was comparable among C282Y heterozygotes, H63D heterozygotes, H63D homozygotes, and patients wild type for HFE. Neither HIC nor HII was significantly associated with posttransplantation survival. However, there appeared to be a trend toward worse survival among patients with higher HII and HIC. It is possible that our sample size lacked sufficient power to discriminate a survival difference between patients with low vs high hepatic iron content. Another factor limiting the interpretation of data examining the effect of HII and HIC on survival is the large biologic variability of HIC and HII within a single liver obtained from needle biopsy samples among patients with iron overload and end-stage liver disease, as previously shown by several groups.10,17,21 We had multiple measurements of HIC in only 11% of samples; therefore, we cannot exclude that our analysis of subgroups based on cutoff values for HII above or below 1.9 and HIC above or below 4000 ␮g/g may not have been sufficiently specific to distinguish between high and low levels of hepatic iron loading. Previous studies examining the effect of HIC and HII as an independent factor on outcome after liver transplantation have provided conflicting results. Brandhagen et al. found that HII ⱖ1.9 was associated with an increased risk of invasive fungal infections and lower Table 6. Causes of Death Among HH and non-HH Patients Figure 4. (A) Kaplan–Meier estimates for survival among patients with HII ⬎1.9 compared with those with HII ⱕ1.9. There was no significant difference in survival between the 2 groups. (B) Kaplan– Meier estimates for survival among patients with hepatic iron concentration (HIC) ⬎70 ␮mol/g dry weight compared with those with HIC ⱕ70 ␮mol/g dry weight. There was no significant difference in survival between the 2 groups.

tients with iron overload and the practice of not obtaining routine liver biopsies in the setting of advanced liver disease, as we have previously described.13 Similarly, iron depletion via phlebotomy was carried out prior to transplantation in only a small minority of the overall study population and in patients subsequently found to have HH. Survival after liver transplantation among patients

HH Cardiovascular only Infection only Cardiovascular and infection Rejection only Infection and rejection Other/unknown Total Non-HH Cardiovascular only Infection only Cardiovascular and infection Rejection only Infection and rejection Other/unknown Total

2 5 1 0 1 7 16

12.50% 31.25% 6.25% 0.00% 6.25% 43.75% 100%

15 14 6 2 0 30 67

15.96% 14.89% 6.38% 2.13% 0.00% 60.64% 100%

NOTE. There was no significant difference in the distribution of causes of death (P ⫽ .19).

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posttransplantation survival.11 However, Stuart et al. reported that HIC ⱖ70-␮mol/g dry weight was not associated with lower posttransplantation survival compared with HIC ⬍70-␮mol/g dry weight.12 We previously reported in a preliminary study that presence of stainable iron in the liver explant was independently associated with a 2.6-fold (95% CI: 1.2–5.6, P ⬍ .01) increased risk of fungal infection after liver transplantation after adjustment for other risk factors for invasive fungal infection, such as hemodialysis, retransplantation, and hospitalization in an intensive care unit.22 Another recent preliminary study from the national UNOS database also suggested that patients carrying a diagnosis of “hemosiderosis” had a decreased survival after liver transplantation and a higher prevalence of infectious or cardiac complications.23 However, this latter study was limited by the lack of HFE genotyping and HIC or HII measurement and did not adjust for confounders that may have influenced posttransplantation survival. In the current study, we found that long-term survival among non-HH patients with hepatic iron overload was also significantly lower compared with the overall survival results for the remaining patients observed at the participating transplantation centers during the time period of the study. However, we would interpret these data with some caution because this analysis did not control for other variables such as age and UNOS status. Nevertheless, these data suggest that hepatic iron overload may diminish survival postliver transplantation even among patients without HH when compared with patients without hepatic iron overload. Comparison of posttransplantation survival in iron-loaded subjects without HH to a matched, control cohort without hepatic iron overload is needed to confirm whether hepatic iron overload in the absence of HH is associated with decreased survival after liver transplantation. In addition, it is possible that patients with hepatic iron overload in the absence of HFE mutations may also have increased infectious and cardiac complications with associated increases in hospital stay, costs, or decreased quality of life when compared with patients without hepatic iron overload that may not be apparent by comparing survival rates alone. There is a substantial body of evidence that iron overload associated with HH is associated with an increased risk of cardiac and infectious complications. Iron deposition in the heart in HH is associated with arrhythmias in up to 40% of patients with cirrhosis4 and is also associated with a range of abnormalities in left ventricular function.24,25 Cardiac iron deposition may also be present in patients with end-stage liver disease and he-

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patic iron overload because of alcoholic liver disease and/or chronic hepatitis C without HH.3,11,15 Bacterial, viral, and fungal infections have been associated with iron overload. Blumberg et al described in 1979 that serum ferritin was associated with the risk of developing chronic infection after exposure to hepatitis B.26 The seroprevalence of both hepatitis B and hepatitis C appears to be increased among patients with HH.27,28 Patients with HH have also been reported to be at increased risk of bacterial infections, in particular Vibrio vulnificus infections.29 Iron overload has also been reported to result in immunodeficiency.30 de Sousa et al have shown a range of abnormalities in T-cell–mediated immunity in patients with hemochromatosis. These include alterations in T-cell repertoires, abnormal CD4:CD8 ratios, and decreased cutaneous hypersensitivity reactions, as well as in vitro studies demonstrating abnormal proliferative responses by peripheral blood mononuclear cells to mitogenic stimuli.30 –34 Thus, it is plausible that iron overload in the setting of immunosuppression after liver transplantation might increase both the risk of infections and serious infections. In conclusion, our study demonstrates decreased survival after liver transplantation among patients with HH characterized by the C282Y homozygous or C282Y/H63D compound heterozygous mutations. Our data also suggest that patients with hepatic iron overload and other HFE genotypes are at increased risk for premature mortality after liver transplantation, although a larger controlled study with matched non–iron-loaded control subjects will be required to confirm these findings. We are unsure of the mechanisms whereby patients with HFE-associated hemochromatosis are at significantly increased risk of premature mortality after liver transplantation, especially because there did not appear to be a significant difference in hepatic iron content between patients with and without HH. We speculate that these differences may be due to differences in extrahepatic storage sites, or that HFE, being an HLA class I protein, may have heretofore unknown functions with regard to the immune response to infection. We believe our findings warrant additional research to improve methods for identification of hepatic iron overload prior to liver transplantation, to characterize better the causes of increased mortality in patients with HH and to examine the feasibility and efficacy of iron depletion prior to liver transplantation in patients with hepatic iron overload.

Appendix 1: The NHTR Study Group Includes the Following Investigators: Baylor University Medical Center: Jeffrey Weinstein, MD; Karla Huang, RN; California Pacific Medical

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Center: Robert G. Gish, MD; Janet Leung; Lahey Clinic: Frederic Gordon, Agnes Trabucco; Mayo Clinic, Rochester, Minnesota: David J. Brandhagen, MD; Heidi Torgerson; Mount Sinai/Cornell University: Michael L. Schilsky, MD; Rush University: Scott J. Cotler, MD; St. Louis University: Bruce R. Bacon, MD; Stanford University: Emmet B. Keeffe, MD; University of California, San Francisco: Nathan Bass, MD, PhD; Lily Leslie Luu; University of Michigan: Robert J. Fontana, MD; University of Nebraska: Timothy McCashland, MD; University of Washington: Kris V. Kowdley, MD; Candi Wines, MPH; Bruce Y Tung, MD; Heather Chewning; Julie Bares; Mary P. Bronner, MD; Stuart Raaka, MS; Del Landicho; Mountain Whisper-Light Statistical Consulting: Nayak Polissar, PhD; Blazej Neradilek, MS.

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13. Cotler SJ, Bronner MP, Press RD, Carlson TH, Perkins JD, Emond MJ, Kowdley KV. End-stage liver disease without hemochromatosis associated with elevated hepatic iron index. J Hepatol 1998; 29:257–262. 14. Ludwig J, Hashimoto E, Porayko MK, Moyer TP, Baldus WP. Hemosiderosis in cirrhosis: a study of 447 native livers. Gastroenterology 1997;112:882– 888. 15. Brandhagen DJ. Liver transplantation for hereditary hemochromatosis. Liver Transpl 2001;7:663– 672. 16. Crawford DH, Fletcher LM, Hubscher SG, Stuart KA, Gane E, Angus PW, Jeffrey GP, McCaughan GW, Kerlin P, Powell LW, Elias EE. Patient and graft survival after liver transplantation for hereditary hemochromatosis: implications for pathogenesis. Hepatology 2004;39:1655–1662. 17. Emond MJ, Bronner MP, Carlson TH, Lin M, Labbe RF, Kowdley KV. Quantitative study of the variability of hepatic iron concentrations. Clin Chem 1999;45:340 –346. 18. Raaka S, Huehnergarth KV, Kowdley KV, Bronner MP. Tissue typing for HFE mutations. Hepatology 2002;35:977–978. 19. Kaplan EL, Meier P. Nonparametric estimation from incomplete observations. J Am Stat Assoc. 1958;53:457– 481. 20. Hosmer DW, Lemeshow S. Applied survival analysis. New York: John Wiley & Sons, 2002 21. Villeneuve JP, Bilodeau M, Lepage R, Cote J, Lefebvre M. Variability in hepatic iron concentration measurement from needlebiopsy specimens. J Hepatol 1996;25:172–177. 22. Limaye AP, Bronner MP, Kowdley KV. Iron excess in the hepatic explant is associated with an increased risk of invasive fungal infection in liver transplant recipients. Hepatology 2001;34:288A. 23. Byrne T, Balan V, Sachdev M, Petre S, Vargas H, Harison ME, Rodriguez-Luna H, Doublas D, Rakela J. Long term outcome of liver transplantation for iron overload: analysis of the Scientific Registry of Transplant Recipients (SRTR) Database. Gastroenterology 2004;126(Suppl 2):A661. 24. Cecchetti G, Binda A, Piperno A, Nador F, Fargion S, Fiorelli G. Cardiac alterations in 36 consecutive patients with idiopathic haemochromatosis: polygraphic and echocardiographic evaluation. Eur Heart J 1991;12:224 –230. 25. Dabestani A, Child JS, Perloff JK, Figueroa WG, Schelbert HR, Engel TR. Cardiac abnormalities in primary hemochromatosis. Ann N Y Acad Sci 1988;526:234 –244. 26. Lustbader ED, Hann HW, Blumberg BS. Serum ferritin as a predictor of host response to hepatitis B virus infection. Science 1983;220:423– 425. 27. Piperno A, Fargion S, D’Alba R, Roffi L, Fracanzani AL, Vecchi L, Failla M, Fiorelli G. Liver damage in Italian patients with hereditary hemochromatosis is highly influenced by hepatitis B and C virus infection. J Hepatol 1992;16:364 –368. 28. Deugnier Y, Battistelli D, Jouanolle H, Guyader D, Gueguen M, Loreal O, Jacquelinet C, Bourel M, Brissot P. Hepatitis B virus infection markers in genetic haemochromatosis. A study of 272 patients. J Hepatol 1991;13:286 –290. 29. Bullen JJ, Spalding PB, Ward CG, Gutteridge JM. Hemochromatosis, iron and septicemia caused by Vibrio vulnificus. Arch Intern Med 1991;151:1606 –1609. 30. de Sousa M, Porto G. The immunological system in hemochromatosis. J Hepatol 1998;28(Suppl 1):1–7. 31. Cardoso C, Porto G, Lacerda R, Resende D, Rodrigues P, Bravo F, Oliveira JC, Justica B, de Sousa M. T-cell receptor repertoire in hereditary hemochromatosis: a study of 32 hemochromatosis patients and 274 healthy subjects. Hum Immunol 2001;62: 488 – 499. 32. Arosa FA, Oliveira L, Porto G, da Silva BM, Kruijer W, Veltman J, de Sousa M. Anomalies of the CD8⫹ T cell pool in haemochromatosis: HLA-A3-linked expansions of CD8⫹CD28- T cells. Clin Exp Immunol 1997;107:548 –554.

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33. Porto G, Vicente C, Teixeira MA, Martins O, Cabeda JM, Lacerda R, Goncalves C, Fraga J, Macedo G, Silva BM, Alves H, Justica B, de Sousa M. Relative impact of HLA phenotype and CD4-CD8 ratios on the clinical expression of hemochromatosis. Hepatology 1997;25:397– 402. 34. Arosa FA, da Silva AJ, Godinho IM, ter Steege JC, Porto G, Rudd CE, de Sousa M. Decreased CD8-p56lck activity in peripheral blood T-lymphocytes from patients with hereditary haemochromatosis. Scand J Immunol 1994;39:426 – 432.

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Received October 22, 2004. Accepted April 20, 2005. Address requests for reprints to: Kris V. Kowdley, MD, Professor of Medicine, University of Washington, Box 356174, Seattle, Washington 98195. e-mail: [email protected]; fax: (206) 5983884. These data were presented in part at the Plenary Session of the Annual Meeting of the American Association for the Study of Liver Diseases, 2001. Supported by NIH grants DK 54698, DK 02957, and DK 38215.