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Hepatocellular carcinoma and focal hepatic glycogenosis after prolonged azathioprine therapy
HUMAN PATHOLOGY
Volume 31, No. 7 (July 2000)
HEPATOCELLULAR CARCINOMA AND FOCAL HEPATIC GLYCOGENOSIS AFTER PROLONGED AZATHIOPRINE THERAPY STE´ PHANE CATTAN, MD, DOMINIQUE WENDUM, MD, OLIVIER CHAZOUILLERES, MD, JACQUES SCHMITZ, MD, AND JEAN-PIERRE GENDRE, MD A 22-year-old woman without predisposing liver disease developed focal hepatic glycogenosis and hepatocellular carcinoma after 6 years of azathioprine therapy for Crohn’s disease. Hepatocellular carcinoma without cirrhosis has previously been described during immunosuppression, but this is the first report of disseminated focal hepatic glycogenosis after long-term azathioprine therapy.
HUM PATHOL 31:874-876. Copyright r 2000 by W.B. Saunders Company Key words: azathioprine, focal hepatic glycogenosis, hepatocellular carcinoma. Abbreviations: HCC, hepatocellular carcinoma; FHG, focal hepatic glycogenosis; G6Pase, glucose-6-phosphatase; PAS, periodic acid-Schiff.
Hepatocellular carcinoma (HCC) without cirrhosis is an unusual complication of immunosuppression, and it occurs almost exclusively in patients with chronic viral hepatitis. Recently, 2 cases of HCC have been reported in patients with long-term azathioprine therapy as the only apparent risk factor.1 We describe the first documented association of focal hepatic glycogenosis (FHG), a potential preneoplastic lesion,2,3 with long-term azathioprine therapy in a patient who developed HCC.
ng/mL. Chemoembolization was performed, and alphafetoprotein level decreased to 8.3 ng/mL. In January 1999 the patient underwent an orthotopic liver transplantation. Histological evaluation of the explanted liver showed 3 nontumoral necrotic nodules. Disseminated FHG persisted 9 months after azathioprine withdrawal.
CASE REPORT A 22-year-old woman was admitted to our hospital in March 1998 for severe abdominal pain. Ileocolonic Crohn’s disease had been diagnosed in 1985 and was well controlled until 1992. Six years before admission, azathioprine was added as a steroid-sparing therapy (2 mg/kg/day). As the disease had been quiescent from 1994 to 1998, azathioprine was withdrawn 1 week before admission. The patient remained on mesalazine 3 g/day. There was no oral contraceptive use. On admission, physical examination showed a right upper quadrant tenderness. A computed tomographic scan (CT-scan) disclosed a 7.5 ⫻ 6 cm partly necrotic mass with otherwise normal-appearing liver parenchyma. Serum alpha-fetoprotein level was 55,000 ng/mL. Hepatitis B surface antigen, hepatitis B surface antibody, hepatitis B core antibody, and hepatitis C virus antibody were all negative. The patient underwent a right hepatic lobectomy. Histological evaluation disclosed a large encapsulated trabecular HCC without surgical margin invasion. There was neither chronic hepatitis, cirrhosis, nor iron overload. Many foci of clear hepatocytes (Fig 1) were observed within the nonneoplastic tissue. Ki-67 immunochemistry was positive in the tumor but negative in all the foci tested. Periodic acid-Schiff (PAS) staining used without and with diastase confirmed glycogen accumulation in the foci (Fig 2). Therefore, as this finding could be suggestive of a carbohydrate metabolism disorder, the diagnosis of type 1a glycogen storage disease was considered. The 5 exons of the glucose-6-phosphatase (G6Pase) gene were sequenced, and no mutation was found. Six months after surgery an HCC recurrence was shown by CT-scan evaluation; alpha-fetoprotein level rose to 5,900
From the Service d’He´pato-Gastroente´rologie, Hoˆpital Rothschild, Service d’Anatomie Pathologique and Service d’He´patoGastroente´rologie, Hoˆpital Saint Antoine, and Service de Pe´diatrie, Hoˆpital Necker Enfants Malades, Paris, France. Address correspondence and reprint requests to Ste´phane Cattan, MD, Service d’He´pato-Gastroente´rologie et Nutrition, Hoˆpital Rothschild, 33, boulevard de Picpus, 75571 Paris Cedex 12. Copyright r 2000 by W.B. Saunders Company 0046-8177/00/3107-0018$10.00/0 doi:10.1053/hupa.2000.7629
DISCUSSION Prolonged immunosuppression predisposes to malignancy.4 An increased risk of non-Hodgkin’s lymphoma and cutaneous squamous cell carcinoma is well established in patients who received long-term azathioprine-based immunosuppressive regimens, but HCC remains exceptional in the absence of hepatitis B, hepatitis C, or chronic liver disease. Such cases have been reported in kidney transplant recipients5,6 and in a patient with Crohn’s disease7 following azathioprine therapy. However, as hepatitis C studies were not available at that time, the hypothesis of an underlying chronic hepatitis C cannot be excluded. Recently, azathioprine was the only identified risk factor in 2 additional patients with HCC.8 Histological examination of the adjacent liver parenchyma was normal in these cases. Thus, it cannot be determined whether HCC was promoted by an azathioprine-induced preneoplastic lesion or not. The mechanism by which azathioprine predisposes to malignancy remains controversial. A decrease in immunological surveillance is supported by the correlation between the incidence of neoplasia and the degree of immunosuppression.1,4,5,8 Azathioprine and 6-mercaptopurine may also have a mutagenic potential.9 Whether or not immunosuppressive therapy increases the risk of malignancy in inflammatory bowel disease patients is much debated. Although published data from 2 referral centers are reassuring, the median durations of immunosuppression reported in these studies were less than 3 years.10,11 As the incidence of malignancy is partly related to immunosuppression duration,8 these results should not be extrapolated to long-term treatment. Focal hepatic glycogenosis is the most likely diagnosis in the present case. The distribution of glycogen accumulation was focal and randomly localized within the lobules. None of the usual clinical or biological features of inherited glycogen storage disease was observed. There was particularly no neutropenia which, in association with Crohn’s disease, could be suggestive of glycogen storage disease.12 However, it was mandatory to rule out this diagnosis, for it predisposes to HCC.13 The absence of mutation within the G6Pase gene coding sequence strongly argues against this possibility in our patient. The cause and significance of FHG are not well known. Experimental studies have shown that the glycogen accumulation is associated with a decreased activity of glycogen phosphorylase and G6Pase within the foci.2 These enzymatic
874
CASE STUDIES
FIGURE 1. Focal hepatic glycogenosis in the non-tumoral liver. (Hematoxylinphloxine-saffron, original magnification ⫻50.)
FIGURE 2. Focus of glycogenated clear hepatocytes. (PAS without (A) and with (B) diastase, original magnification ⫻200.)
disorders may be associated with a focal overexpression of the main intracellular substrate of the insulin receptor.14 Glycogenated foci have also been reported in children with urea cycle disorders responsible for Reye’s-like syndrome, such as ornithine transcarbamoylase deficiency, carbamyl-phosphate synthase I deficiency, and hyperammonemia-hyperornithinemiahomocitrullinuria.15 On the other hand, there is increasing evidence that glycogenated foci are preneoplastic lesions. Both histological and biochemical similarities exist between FHG and lesions observed during drug-induced hepatocarcinogenesis, and the presence of FHG within nonmalignant hepatocellular nodules of cirrhotic livers could predict their malignant transformation.3 Of note, the association between FHG and azathioprine has not been reported yet, and this association may be coincidental. Actually, FHG has already been described in both cirrhotic and non-cirrhotic livers bearing HCC.2 However, we believe that the age of our patient, the absence of predisposing liver disease and especially the disseminated pattern of FHG argue in favor of a pathogenic link between long-term azathioprine therapy and FHG in the present case. The persistence of FHG on the transplant, despite azathioprine withdrawal, further indicate an acquired genetic preneoplastic alteration. Subsequently, FHG may have promoted the development of HCC. One possible, but unproven, mechanistic association between azathioprine and FHG may involve an hepatotropic effect of the drug. Azathioprine has been shown to increase hepatocyte turnover in an experimental study,16 and a similar effect could be responsible for the high proliferation index observed within glycogenated foci.2 In conclusion, the role of azathioprine in the occurrence of FHG and HCC cannot be determined from the single case presented here. However, our report provides additional arguments in favor of a pathogenic link between FHG and HCC. Further studies are needed to confirm the suggested role of prolonged azathioprine therapy in the occurence of FHG.
875
HUMAN PATHOLOGY
Volume 31, No. 7 (July 2000)
REFERENCES 1. Saeian K, Franco J, Komorowski RA, et al: Hepatocellular carcinoma after renal transplantation in the absence of cirrhosis or viral hepatitis: A case series. Liver Transpl Surg 5:46-49, 1999 2. Bannasch P, Jahn UR, Hacker HJ, et al: Focal hepatic glycogenosis: A putative preneoplastic lesion associated with neoplasia and cirrhosis in explanted human liver. Int J Oncol 10:261-268, 1997 3. Terasaki S, Kaneko S, Kobayashi K, et al: Histological features predicting malignant transformation of nonmalignant hepatocellular nodules: A prospective study. Gastroenterology 115:1216-1222, 1998 4. Xerri L, Payan MJ, Choux R, et al: An exceptional 18-year follow-up after cardiac transplantation. How can malignancies occur during immunosuppressive therapy? Cancer 63:1697-1699, 1989 5. Gruber S, Dehner LP, Simmons RL: De novo hepatocellular hepatocarcinoma without chronic liver disease but with 17 years of azathioprine immunosuppression. Transplantation 43:597-600, 1987 6. Gardner BP, Evans DB: Primary hepatocellular carcinoma arising in a renal transplant recipient with polycystic disease. Postgrad Med J 59:120-121, 1983 7. Lee FL, Murray SM, Prior J, et al: Primary liver cell cancer occuring in association with Crohn’s disease treated with prednisolone and azathioprine. Hepatogastroenterology 30:188, 1983
8. Jensen P, Hansen S, Moller B, et al: Skin cancer in kidney and heart transplant and different long-term immunosuppressive therapy regimens. J Am Acad Dermatol 40:177-186, 1999 9. Kwong YL, Au WY, Liang RH: Acute myeloid leukemia after azathioprine treatment for autoimmune diseases: Association with ⫺7/7q-. Cancer Genet Cytogenet 104:94-97, 1998 10. Connell WR, Kamm MA, Dickson M, et al: Long-term neoplasia risk after azathioprine treatment in inflammatory bowel disease. Lancet 343:12491252, 1994 11. Present DH, Meltzer Sj, Krumholz MP, et al: 6-mercaptopurine in the management of inflammatory bowel disease: Short and long term toxicity. Ann Intern Med 111:641-649, 1989 12. Roe T, Schonfeld N, Thomas D, et al: Regional enteritis and glycogen storage disease type Ib. Lancet 1:1077, 1984 13. Bianchi L: Glycogen storage disease I and hepatocellular tumours. Eur J Pediatr 152:S63-S70, 1993 (suppl 1) 14. Nehrbass D, Klimek F, Bannasch P: Overexpression of insulin receptor substrate-1 emerges early in hepatocarcinogenesis and elicits preneoplastic hepatic glycogenosis. Am J Pathol 152:341-345, 1998 15. Badizadegan K, Perez-Atayde AR: Focal glycogenosis of the liver in disorders of ureagenesis: Its occurrence and diagnostic significance. Hepatology 26:365-373, 1997 16. Arber N, Zajicek G, Nordenberg J, et al: Azathioprine treatment increases hepatocyte turnover. Gastroenterology 101:1083-1086, 1991
876
Volume 31, No. 7 (July 2000)
HEPATOCELLULAR CARCINOMA AND FOCAL HEPATIC GLYCOGENOSIS AFTER PROLONGED AZATHIOPRINE THERAPY STE´ PHANE CATTAN, MD, DOMINIQUE WENDUM, MD, OLIVIER CHAZOUILLERES, MD, JACQUES SCHMITZ, MD, AND JEAN-PIERRE GENDRE, MD A 22-year-old woman without predisposing liver disease developed focal hepatic glycogenosis and hepatocellular carcinoma after 6 years of azathioprine therapy for Crohn’s disease. Hepatocellular carcinoma without cirrhosis has previously been described during immunosuppression, but this is the first report of disseminated focal hepatic glycogenosis after long-term azathioprine therapy.
HUM PATHOL 31:874-876. Copyright r 2000 by W.B. Saunders Company Key words: azathioprine, focal hepatic glycogenosis, hepatocellular carcinoma. Abbreviations: HCC, hepatocellular carcinoma; FHG, focal hepatic glycogenosis; G6Pase, glucose-6-phosphatase; PAS, periodic acid-Schiff.
Hepatocellular carcinoma (HCC) without cirrhosis is an unusual complication of immunosuppression, and it occurs almost exclusively in patients with chronic viral hepatitis. Recently, 2 cases of HCC have been reported in patients with long-term azathioprine therapy as the only apparent risk factor.1 We describe the first documented association of focal hepatic glycogenosis (FHG), a potential preneoplastic lesion,2,3 with long-term azathioprine therapy in a patient who developed HCC.
ng/mL. Chemoembolization was performed, and alphafetoprotein level decreased to 8.3 ng/mL. In January 1999 the patient underwent an orthotopic liver transplantation. Histological evaluation of the explanted liver showed 3 nontumoral necrotic nodules. Disseminated FHG persisted 9 months after azathioprine withdrawal.
CASE REPORT A 22-year-old woman was admitted to our hospital in March 1998 for severe abdominal pain. Ileocolonic Crohn’s disease had been diagnosed in 1985 and was well controlled until 1992. Six years before admission, azathioprine was added as a steroid-sparing therapy (2 mg/kg/day). As the disease had been quiescent from 1994 to 1998, azathioprine was withdrawn 1 week before admission. The patient remained on mesalazine 3 g/day. There was no oral contraceptive use. On admission, physical examination showed a right upper quadrant tenderness. A computed tomographic scan (CT-scan) disclosed a 7.5 ⫻ 6 cm partly necrotic mass with otherwise normal-appearing liver parenchyma. Serum alpha-fetoprotein level was 55,000 ng/mL. Hepatitis B surface antigen, hepatitis B surface antibody, hepatitis B core antibody, and hepatitis C virus antibody were all negative. The patient underwent a right hepatic lobectomy. Histological evaluation disclosed a large encapsulated trabecular HCC without surgical margin invasion. There was neither chronic hepatitis, cirrhosis, nor iron overload. Many foci of clear hepatocytes (Fig 1) were observed within the nonneoplastic tissue. Ki-67 immunochemistry was positive in the tumor but negative in all the foci tested. Periodic acid-Schiff (PAS) staining used without and with diastase confirmed glycogen accumulation in the foci (Fig 2). Therefore, as this finding could be suggestive of a carbohydrate metabolism disorder, the diagnosis of type 1a glycogen storage disease was considered. The 5 exons of the glucose-6-phosphatase (G6Pase) gene were sequenced, and no mutation was found. Six months after surgery an HCC recurrence was shown by CT-scan evaluation; alpha-fetoprotein level rose to 5,900
From the Service d’He´pato-Gastroente´rologie, Hoˆpital Rothschild, Service d’Anatomie Pathologique and Service d’He´patoGastroente´rologie, Hoˆpital Saint Antoine, and Service de Pe´diatrie, Hoˆpital Necker Enfants Malades, Paris, France. Address correspondence and reprint requests to Ste´phane Cattan, MD, Service d’He´pato-Gastroente´rologie et Nutrition, Hoˆpital Rothschild, 33, boulevard de Picpus, 75571 Paris Cedex 12. Copyright r 2000 by W.B. Saunders Company 0046-8177/00/3107-0018$10.00/0 doi:10.1053/hupa.2000.7629
DISCUSSION Prolonged immunosuppression predisposes to malignancy.4 An increased risk of non-Hodgkin’s lymphoma and cutaneous squamous cell carcinoma is well established in patients who received long-term azathioprine-based immunosuppressive regimens, but HCC remains exceptional in the absence of hepatitis B, hepatitis C, or chronic liver disease. Such cases have been reported in kidney transplant recipients5,6 and in a patient with Crohn’s disease7 following azathioprine therapy. However, as hepatitis C studies were not available at that time, the hypothesis of an underlying chronic hepatitis C cannot be excluded. Recently, azathioprine was the only identified risk factor in 2 additional patients with HCC.8 Histological examination of the adjacent liver parenchyma was normal in these cases. Thus, it cannot be determined whether HCC was promoted by an azathioprine-induced preneoplastic lesion or not. The mechanism by which azathioprine predisposes to malignancy remains controversial. A decrease in immunological surveillance is supported by the correlation between the incidence of neoplasia and the degree of immunosuppression.1,4,5,8 Azathioprine and 6-mercaptopurine may also have a mutagenic potential.9 Whether or not immunosuppressive therapy increases the risk of malignancy in inflammatory bowel disease patients is much debated. Although published data from 2 referral centers are reassuring, the median durations of immunosuppression reported in these studies were less than 3 years.10,11 As the incidence of malignancy is partly related to immunosuppression duration,8 these results should not be extrapolated to long-term treatment. Focal hepatic glycogenosis is the most likely diagnosis in the present case. The distribution of glycogen accumulation was focal and randomly localized within the lobules. None of the usual clinical or biological features of inherited glycogen storage disease was observed. There was particularly no neutropenia which, in association with Crohn’s disease, could be suggestive of glycogen storage disease.12 However, it was mandatory to rule out this diagnosis, for it predisposes to HCC.13 The absence of mutation within the G6Pase gene coding sequence strongly argues against this possibility in our patient. The cause and significance of FHG are not well known. Experimental studies have shown that the glycogen accumulation is associated with a decreased activity of glycogen phosphorylase and G6Pase within the foci.2 These enzymatic
874
CASE STUDIES
FIGURE 1. Focal hepatic glycogenosis in the non-tumoral liver. (Hematoxylinphloxine-saffron, original magnification ⫻50.)
FIGURE 2. Focus of glycogenated clear hepatocytes. (PAS without (A) and with (B) diastase, original magnification ⫻200.)
disorders may be associated with a focal overexpression of the main intracellular substrate of the insulin receptor.14 Glycogenated foci have also been reported in children with urea cycle disorders responsible for Reye’s-like syndrome, such as ornithine transcarbamoylase deficiency, carbamyl-phosphate synthase I deficiency, and hyperammonemia-hyperornithinemiahomocitrullinuria.15 On the other hand, there is increasing evidence that glycogenated foci are preneoplastic lesions. Both histological and biochemical similarities exist between FHG and lesions observed during drug-induced hepatocarcinogenesis, and the presence of FHG within nonmalignant hepatocellular nodules of cirrhotic livers could predict their malignant transformation.3 Of note, the association between FHG and azathioprine has not been reported yet, and this association may be coincidental. Actually, FHG has already been described in both cirrhotic and non-cirrhotic livers bearing HCC.2 However, we believe that the age of our patient, the absence of predisposing liver disease and especially the disseminated pattern of FHG argue in favor of a pathogenic link between long-term azathioprine therapy and FHG in the present case. The persistence of FHG on the transplant, despite azathioprine withdrawal, further indicate an acquired genetic preneoplastic alteration. Subsequently, FHG may have promoted the development of HCC. One possible, but unproven, mechanistic association between azathioprine and FHG may involve an hepatotropic effect of the drug. Azathioprine has been shown to increase hepatocyte turnover in an experimental study,16 and a similar effect could be responsible for the high proliferation index observed within glycogenated foci.2 In conclusion, the role of azathioprine in the occurrence of FHG and HCC cannot be determined from the single case presented here. However, our report provides additional arguments in favor of a pathogenic link between FHG and HCC. Further studies are needed to confirm the suggested role of prolonged azathioprine therapy in the occurence of FHG.
875
HUMAN PATHOLOGY
Volume 31, No. 7 (July 2000)
REFERENCES 1. Saeian K, Franco J, Komorowski RA, et al: Hepatocellular carcinoma after renal transplantation in the absence of cirrhosis or viral hepatitis: A case series. Liver Transpl Surg 5:46-49, 1999 2. Bannasch P, Jahn UR, Hacker HJ, et al: Focal hepatic glycogenosis: A putative preneoplastic lesion associated with neoplasia and cirrhosis in explanted human liver. Int J Oncol 10:261-268, 1997 3. Terasaki S, Kaneko S, Kobayashi K, et al: Histological features predicting malignant transformation of nonmalignant hepatocellular nodules: A prospective study. Gastroenterology 115:1216-1222, 1998 4. Xerri L, Payan MJ, Choux R, et al: An exceptional 18-year follow-up after cardiac transplantation. How can malignancies occur during immunosuppressive therapy? Cancer 63:1697-1699, 1989 5. Gruber S, Dehner LP, Simmons RL: De novo hepatocellular hepatocarcinoma without chronic liver disease but with 17 years of azathioprine immunosuppression. Transplantation 43:597-600, 1987 6. Gardner BP, Evans DB: Primary hepatocellular carcinoma arising in a renal transplant recipient with polycystic disease. Postgrad Med J 59:120-121, 1983 7. Lee FL, Murray SM, Prior J, et al: Primary liver cell cancer occuring in association with Crohn’s disease treated with prednisolone and azathioprine. Hepatogastroenterology 30:188, 1983
8. Jensen P, Hansen S, Moller B, et al: Skin cancer in kidney and heart transplant and different long-term immunosuppressive therapy regimens. J Am Acad Dermatol 40:177-186, 1999 9. Kwong YL, Au WY, Liang RH: Acute myeloid leukemia after azathioprine treatment for autoimmune diseases: Association with ⫺7/7q-. Cancer Genet Cytogenet 104:94-97, 1998 10. Connell WR, Kamm MA, Dickson M, et al: Long-term neoplasia risk after azathioprine treatment in inflammatory bowel disease. Lancet 343:12491252, 1994 11. Present DH, Meltzer Sj, Krumholz MP, et al: 6-mercaptopurine in the management of inflammatory bowel disease: Short and long term toxicity. Ann Intern Med 111:641-649, 1989 12. Roe T, Schonfeld N, Thomas D, et al: Regional enteritis and glycogen storage disease type Ib. Lancet 1:1077, 1984 13. Bianchi L: Glycogen storage disease I and hepatocellular tumours. Eur J Pediatr 152:S63-S70, 1993 (suppl 1) 14. Nehrbass D, Klimek F, Bannasch P: Overexpression of insulin receptor substrate-1 emerges early in hepatocarcinogenesis and elicits preneoplastic hepatic glycogenosis. Am J Pathol 152:341-345, 1998 15. Badizadegan K, Perez-Atayde AR: Focal glycogenosis of the liver in disorders of ureagenesis: Its occurrence and diagnostic significance. Hepatology 26:365-373, 1997 16. Arber N, Zajicek G, Nordenberg J, et al: Azathioprine treatment increases hepatocyte turnover. Gastroenterology 101:1083-1086, 1991
876