Increased Hepatic Iron Deposition Resulting From Treatment of Chronic Hepatitis C With Ribavirin

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1 Anatomic Pathology / INCREASED HEPATIC IRON DEPOSITION ASSOCIATED WITH RIBAVIRIN THERAPY Increased Hepatic Iron Deposition Resulting From Treatment of Chronic Hepatitis C With Ribavirin M. Isabel Fiel, MD, 1 Thomas D. Schiano, MD, 2 Maria Guido, MD, 1 Swan N. Thung, MD, 1 Karen L. Lindsay, MD, 3 Gary L. Davis, MD, 4 James H. Lewis, MD, 5 Leonard B. Seeff, MD, 6 and Henry C. Bodenheimer, Jr, MD 2 Key Words: Hemosiderosis; Viral hepatitis; Hepatitis C virus, Interferon Abstract Increased levels of hepatic iron may impair the response of patients with chronic hepatitis C to treatment with interferon-alfa, but combination therapy with ribavirin has demonstrated efficacy in the treatment of hepatitis C. When used alone or with interferon-alfa, ribavirin may cause a dose-dependent reversible hemolytic anemia. We compared the extent and cellular localization of iron deposition in liver tissue from biopsy specimens obtained before and after 36 weeks of therapy with ribavirin or placebo for 59 patients with chronic hepatitis C. Paired slides were available for review from 26 ribavirin and 27 placebo recipients. Iron deposition was assessed using coded slides stained with Perls Prussian blue and was semiquantitated in hepatocytes, Kupffer cells, and areas of fibrosis. The overall iron score fell by 0.96 in the placebo group and increased 1.69 in the ribavirin recipients. Iron was deposited mainly in hepatocytes; the hepatocyte iron score increased from 2.19 to 3.81 in the ribavirin group. The amount of iron staining in Kupffer cells declined in the placebo group and increased slightly in the ribavirin group. Iron changes in areas of fibrosis were minor and did not differ between groups. Increased total hepatic iron deposition occurred during a 9-month course of ribavirin. Ribavirin-associated hemolysis deposits iron preferentially in hepatocytes. This increased deposition of hepatic iron does not seem to affect the biochemical or histologic response to ribavirin therapy but may have implications for hepatocyte susceptibility to future injury. Chronic hepatitis C virus (HCV) infection is a major causative factor responsible for the development of cirrhosis. Significant iron deposition occurs in cirrhosis secondary to any cause, but especially in HCV and alcohol-related chronic liver disease. 1 Increased iron deposition, possibly related to the release of iron from injured hepatocytes, also is observed in patients with HCV who do not have cirrhosis. 2 High levels of hepatic iron may negatively influence the response to interferon-alfa therapy. 3-5 Lower hepatic iron concentration favors a beneficial response to treatment. 3 Phlebotomy decreases serum aminotransferase activities in patients with HCV and may improve the response to interferon-alfa in some patients. 6 Ribavirin, an oral purine nucleoside analog, has a broad spectrum of antiviral activity. 7,8 This drug, however, does not lower hepatitis C viral titers When used in combination with interferon-alfa in the treatment of HCV, the rates of normalization of aminotransferase values, viral eradication, and histologic improvement are higher than when interferon-alfa is used alone One major adverse effect of ribavirin is a dose-dependent reversible hemolysis. 8,9,15,17-20 Chronic intravascular hemolysis typically leads to increased reticuloendothelial cell iron deposition with little increase in hepatocyte iron. However, ribavirin-induced hemolysis is postulated to occur extravascularly within the reticuloendothelial system. 21,22 Hepatic iron accumulation may predispose liver cells to injury from oxidative stress. In addition, a beneficial response to interferon-alfa may be blunted. 15 We evaluated stainable hepatic iron before and after ribavirin therapy of HCV. The changes noted were correlated with the clinical Am J Clin Pathol 2000;113:

2 Fiel et al / INCREASED HEPATIC IRON DEPOSITION ASSOCIATED WITH RIBAVIRIN THERAPY response to ribavirin as measured by alanine aminotransferase (ALT) activity. Results are expressed as mean ± SD with range (minimum and maximum). Materials and Methods This project was part of a multicenter, double-blind, randomized, controlled trial comparing ribavirin and placebo therapy in 59 patients with HCV. 12 Twenty-nine patients received ribavirin at a dose of 1,200 mg/d, and 30 patients received placebo for a duration of 36 weeks. Liver biopsies were performed within 6 months before enrollment and at the end of therapy. Patients were not taking iron supplements and had no history of hemochromatosis or porphyria cutanea tarda. No patient had a history of chronic or hemolytic anemia, and none were using alcohol. Fifty-three paired slides were available and coded, 26 from the ribavirin group and 27 from the placebo group. Perls Prussian blue staining for trivalent iron was applied on 5-µm-thick sections of the liver biopsy specimens. Two pathologists (M.I.F. and M.G.), blinded to the identity of the treatment group and to whether biopsy specimens were obtained before or after treatment, reviewed the slides. Quantitative and qualitative scores for hepatic iron deposition were determined by consensus. Stainable iron was assessed by using the semiquantitative grading system proposed by Tirmann-Schmelzer with modifications. 23 Brissot and coworkers 23 demonstrated this method to be reflective of hepatic iron concentration because it takes into account the different iron storage areas of the hepatic lobule. Briefly, hepatocyte iron, Kupffer cell iron, and areas of fibrosis were assessed and given scores ranging from 0 to 4 arriving at an elementary score A. The hepatocyte score is multiplied by a coefficient of 3, and all 3 scores are then cumulated (score B). The scores are based on the number and percentage of cells containing iron and the areas of iron deposits. The histologic iron estimation included determination of the intensity of iron load according to the following scores: 1, sparse, fine granules; 2, patchy, fine granules; 3, diffuse, fine granules; and 4, diffuse, coarse granules. Total iron scores (score B), hepatocyte iron scores, and Kupffer cell iron scores before and after treatment were compared within each group and then between the 2 treatment groups by using Fisher exact test for statistical analysis. The ALT response at the end of therapy was correlated with change in iron scores. The definition of a positive therapeutic response was normalization of ALT activity at the end of therapy. Other parameters evaluated included mean hemoglobin and reticulocyte count before and after treatment. Results Hemoglobin levels decreased by a mean of 13% (range, 0-4 g/dl [0-40 g/l]) in the ribavirin treated group. A decline in the hemoglobin level of more than 2 g/dl (20 g/l) occurred in 79% of patients receiving ribavirin; a decline of 4 g/dl (40 g/l) or more was observed in 21% of patients. No appreciable decrease in the hemoglobin level was noted in the placebo group. The reticulocyte count increased by a mean of 6.9% at the end of treatment in the ribavirin-treated patients. The semiquantitative evaluation of iron deposition in stained slides is shown in Table 1. The overall iron score at week 0 in the ribavirin-treated group was 3.04 ± 3.75 (range, 0-12), and the overall iron score at week 36 was 4.73 ± 4.03 (range, 0-12). The difference in total iron scores increased from week 0 to week 36 by In the placebo group, the overall iron score at week 0 was 3.74 and at week 36 was 2.77, demonstrating a slight decline of 0.97 by the conclusion of the study. The hepatocyte iron score in the ribavirin-treated group at the time of enrollment was 2.19 ± 3.75 (range, 0-2), and at week 36, the score was 3.81 ± 4.03 (range, 0-12) (Table 1). The difference between week 0 and week 36 was In the placebo group, the hepatocyte iron score at week 0 was 2.56 ± 4.07 (range, 0-12), and at week 36, the hepatocyte iron score was 2.0 ± 3.43 (range, 0-11). The score of Kupffer cell iron deposition in the ribavirin treated group at baseline was 0.81 ± 1.06 (range, 0-3), and at week 36, it was 0.92 ± 1.06 (range, 0-3). In the placebo group, the Kupffer cell iron score at week 0 was 0.89 ± 0.97 (range, 0-3), and at week 36, the score was 0.52 ± 0.70 (range, 0-2) (Table 1). Image 1 and Image 2 demonstrate the change in iron deposition before and after treatment. Normalization of ALT at week 36 did not correlate with the total amount of iron deposition nor change in iron scores Table 2 and Table 3. Discussion HCV infection often progresses to chronic hepatitis, cirrhosis, and hepatocellular carcinoma. At present, interferon-alfa is the only therapy approved for treatment of HCV infection. 24 However, approximately 50% of those treated have no response, and of the patients who show response, 50% to 80% will experience relapse within 6 months of discontinuation of therapy. 25 Combined treatment with interferon-alfa and ribavirin has shown promise for normalizing serum aminotransferase values 36 Am J Clin Pathol 2000;113:35 39

3 Anatomic Pathology / ORIGINAL ARTICLE Image 1 Liver biopsy specimen obtained before ribavirin treatment. Fine granules of iron deposits are evident in Kupffer cells and hepatocytes. Hepatocyte score A of 1 and Kupffer cell score of 1 (Perls Prussian blue, original magnification 160). Image 2 Liver biopsy specimen obtained after 36 weeks of ribavirin therapy. Note the fine and coarse granules present mostly in hepatocytes. Arrows point to occasional Kupffer cells containing iron granules. This biopsy specimen was given a hepatocyte score of 3 and Kupffer cell score of 2 (Perls Prussian blue, original magnification 160). Table 1 Summary of Quantitative and Qualitative Assessment of Hepatic Iron Deposition Ribavirin Group Placebo Group P Week 0 Total score Hepatocytes ( 3) Kupffer cells Areas of fibrosis Week 36 Total score Hepatocytes ( 3) Kupffer cells Areas of fibrosis Change during treatment Total score <.02 Hepatocytes <.02 Kupffer cells <.05 Areas of fibrosis and decreasing the degree of histologic damage in patients with HCV. 13,14,26,27 Distribution of iron in the liver and the amount of iron deposition may have an influence on the patient s response to interferon-alfa therapy. 3-5 In a study by Barton et al, 4 total hepatic iron scores were higher in patients with incomplete response. In addition, Olynyk et al 3 reported that hepatic iron concentration has an influence on the response to therapy for chronic hepatitis C with interferon-alfa. Ribavirin has been used for many years as a treatment for respiratory syncytial virus infection. 28 Its predominant adverse effect is dose-dependent hemolysis and resulting anemia Declines in hematocrit may range from 10% to 13% ( ) with a compensatory increase in reticulocyte counts 8,9 and an increase in total serum bilirubin. 10 The mechanism of this hemolysis is thought to relate to the RBC accumulation of ribavirin triphosphate. 21,22 The increased hepatic iron deposition likely results from uptake of iron released from destroyed RBCs. Di Bisceglie and coworkers 2 also have postulated that ribavirin may cause increased iron absorption from the gastrointestinal tract. In their study of ribavirin effects, iron staining was noted to increase in hepatocytes and Kupffer cells after ribavirin treatment, but there was no change in Am J Clin Pathol 2000;113:

4 Fiel et al / INCREASED HEPATIC IRON DEPOSITION ASSOCIATED WITH RIBAVIRIN THERAPY Table 2 Correlation of Alanine Aminotransferase (ALT) Response With Change in Iron Scores in Ribavirin-Treated Patients Response * Number Decreased Score No Change Increased Score Yes No * Yes indicates a normalization of ALT values; no, a lack of normalization of ALT at week 36. Table 3 Correlation of Alanine Aminotransferase (ALT) Response With Total Iron Scores at Week 36 Response * Number Score 0 Score 1-6 Score >6 Yes No * Yes indicates a normalization of ALT values; no, a lack of normalization of ALT at week 36. the relative cellular distribution of iron. Iron released intravascularly due to hemolysis likely would be taken up mainly by Kupffer cells. In the present study and work by others, 2 iron staining also was seen within hepatocytes. The degree of increased iron deposition seems greater within hepatocytes than other liver cells. Ferritin that accumulates in Kupffer cells as a consequence of RBC uptake and destruction subsequently may be transferred to adjacent hepatocytes. An elegant study done by Sibille et al 29 showed iron, in the form of ferritin, being released by the phagocytosing Kupffer cells and rapidly taken up by hepatocytes. We believe this may explain why, in the present study, more abundant iron deposition was found in hepatocytes. Piperno and colleagues 30 postulated that iron overload may contribute to persistent HCV infection by supplying the virus with essential nutrients and by producing immune alteration that impairs recovery. This may become important over time in ribavirin-treated patients if hepatic iron deposition continues to increase. 2 Hepatic iron stores increased significantly in patients treated with ribavirin compared with those treated with placebo. This increase was demonstrated by using an evaluation of histologic hepatic iron that correlates with the hepatic iron concentration. 2,23 Change in ALT levels during ribavirin therapy was not related to iron deposition before treatment or to liver iron levels after therapy. The long-term effects of increased hepatic iron stores secondary to ribavirin treatment remain to be evaluated. As longer courses of treatment and long-term maintenance therapy with ribavirin alone or in combination with interferon-alfa are contemplated, excessive amounts of iron in the liver could limit the usefulness of ribavirin. Accumulated iron deposition within the liver might reach excessive levels during prolonged treatment, especially in patients with high preexisting concentrations of hepatic iron. From the 1 The Lillian and Henry M. Stratton-Hans Popper Department of Pathology and the 2 Division of Liver Diseases, Department of Medicine, The Mount Sinai Medical Center of the City University of New York, New York, NY; the 3 Division of Gastrointestinal and Liver Diseases, Department of Medicine, University of Southern California, Los Angeles, CA; the 4 Section of Hepatobiliary Diseases, Department of Medicine, University of Florida, Gainesville; the 5 Division of Liver Diseases, Division of Gastroenterology, Department of Medicine, Georgetown University Medical Center, Washington, DC; and the 6 National Institutes of Health, Bethesda, MD. Presented at The American Association for the Study of Liver Diseases Annual Meeting, Chicago, IL, November Published in abstract form in Hepatology. 1995;22:291A. Address reprint requests to Dr Bodenheimer: Division of Liver Diseases, Dept of Medicine, The Mount Sinai Medical Center, One Gustave Levy Place, New York, NY References 1. Bacon BR. Diagnosis and management of hemochromatosis. Gastroenterology. 1997;13: Di Bisceglie AM, Bacon BR, Kleiner DE, et al. Increase in hepatic iron stores following prolonged therapy with ribavirin in patients with chronic hepatitis C. J Hepatol. 1994;21: Olynyk JK, Reddy KR, Di Bisceglie AM, et al. Hepatic iron concentration as a predictor of response to interferon alfa therapy in chronic hepatitis C. Gastroenterology. 1995;108: Barton AL, Banner BF, Cable EE, et al. Distribution of iron in the liver predicts the response of chronic hepatitis C infection to interferon therapy. Am J Clin Pathol. 1995;103: Clemente MG, Congia M, Lai ME, et al. Effect of iron overload on the response to recombinant interferon-alfa treatment in transfusion-dependent patients with thalassemia major and chronic hepatitis C. J Pediatr. 1994;125: Am J Clin Pathol 2000;113:35 39

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