Chronic hepatitis B surface antigen-negative hepatitis after treatment of malignancy Flavio Rossetti, MD, Simone C e s a r o , MD, Patrizia Pizzocchero, MD, Paolo C a d r o b b i , MD, Maria G u i d o , MD, a n d Luigi Z a n e s c o , MD From the Department of Pediatrics, Division of Hemato-Oncology, Division of infectious Diseases, and Istituto di Anatomia Patologica, Cattedra di Istochimica Patologica, University of Padova, Italy We reviewed the records of all patients with a diagnosis of m a l i g n a n c y who were treated at our center and who had not had c h e m o t h e r a p y for at least 18 months, to assess the p r e v a l e n c e of chronic hepatitis B surface antigen (HBsAg)-negative hepatitis, to assess the p r e v a l e n c e of a marker of hepatitis C virus infection, and to determine the severity of chronic liver disease. Of 557 e l i g i b l e patients, 38 (6.8%) had chronic HBsAg-negative hepatitis. Of these 38 patients, 20 (52.6%) had a marker of hepatitis C virus infection. The p r e v a l e n c e of chronic HBsAgn e g a t i v e hepatitis was higher in patients previously treated for leukemia than in patients treated for another m a l i g n a n c y (11.8% vs 4.6%; p = 0.004). The liver biopsy revealed chronic active hepatitis or cirrhosis or both in 8 (28%) of 28 patients with clinical chronic HBsAg-negative hepatitis. Four patients without hepatitis C virus infection who underwent liver biopsy had hepatitis B virus antigen in the liver, confirmed by immunohistochemistry studies. One patient uninfected with hepatitis C virus had hemochromatosis. We c o n c l u d e that infection with hepatitis C virus was the major cause of chronic HBsAg-negative hepatitis in pediatric patients previously treated for malignancy; the cause remained unidentified in 30% of the patients. (J PEDIATR1992;121:39-43) Non-A, non-B hepatitis is one of the most frequent forms of viral hepatitis, and accounts for 80% to 90% of posttransfusion hepatitis. 1 In the pediatric hemato-oncology setting, chronic hepatitis with detectable hepatitis B surface antigen has been the major cause of chronic liver disease, especially in long-term survivors.2, 3 Higher sensitivity of blood-product screening methods for infection with hepatitis B virus and better environmental management have decreased the incidence of HBV infection in these patients4; this accounts for the increasing prevalence of acute and chronic non-A, non-B hepatitis. 5, 6 We reviewed the records of all patients who had malignancy diagnosed and treated at our center and who had had no chemotherapy for at least 18 months, to assess the preySubmitted for publicationMarch 25, 1991;accepted Feb. 13, 1992. Reprint requests: Flavio Rossetti, MD, Department of Pediatrics, Divisionof Hemato-Oncology, Via Giustiniani, 3, 35128 Padova, Italy. 9/20/37100
alence of chronic HBsAg-negative hepatitis, to assess positivity for a marker of infection with hepatitis C virus, and to determine the severity of liver disease in this group of patients. ALT ELISA HBcAg HBsAg HBV HCV
Alanine aminotransferase Enzyme-linked immunosorbent assay HBV core antigen HBV surface antigen Hepatitis B virus Hepatitis C virus
METHODS Patients. From 1967 through June 30, 1990, a total of 557 patients were treated for malignancy at the University of Padova Department of Pediatrics, Division of Hemato-Oncology, and were at least 18 months from the completion of treatment for malignancy. Their medical records were reviewed for a history of elevated serum aminotransferase values lasting more than 12 months from the completion of
39
40
R o s s e t t i et al.
The Journal o f Pediatrics July 1992
Figure. Iron stores in periportal hepatocytes. Some portal tract macrophages are heavily laden; Perls test result is positive. (Hematoxylin-eosin stain; • chemotherapy, to identify patients with chronic HBsAgnegative hepatitis. The patients who had elevated serum aminotransferase values (normal range, 0 to 50 U / L [0.85 mmol/L]) only during the year after the chemotherapy was discontinued were not included in the study, because of the likely influence of hepatotoxic effects of chemotherapy. 4, 7 We defined chronic HBsAg-negative hepatitis as a persistent elevation of alanine aminotransferase level for more than 12 months after the cessation of chemotherapy, and with no evidence of HBsAg positivity. Virologic markers. Markers for HBV were studied at least yearly in each patient who entered the study. HBsAg, hepatitis B e antigen, anti-HBsAg, anti-hepatitis B e antigen and anti-hepatitis B core antigen antibodies were tested by radioimmunoassay or by enzymatic immunoassay (Abbott Laboratories, Diagnostic Division, Latina, Italy, or Sorin Biomedica SPA, Saluggia, italy). Anti-HCV antibody was measured by the Ortho HCV enzyme-linked immunosorbent assay (Ortho Diagnostic Systems, Milan, Italy) in patients with chronic HBsAg-negative hepatitis. In accordance with instructions provided by the manufacturer, the test was done in microwell plates by an automated procedure. Each plate included one blank and three negative and two positive control specimens. The cutoff value was calculated by adding 0.400 to the mean value for negative control specimens. The specimens were considered to be definitely reactive if adsorbance values above the cutoff point were confirmed on retesti~i~Six sera from patients with autoantibodies were retested with a recombinant immunoblot test (HCV RIBA II test, Chiron Corp., Emeryville, Calif.) specific for HCV antibodies. Assessment of hepatic disease. After the completion of
treatment for malignancy, the following tests were performed, at least once, to rule out other causes of chronic liver disease in the patients with chronic HBsAg-negative hepatitis: serum cq-antitrypsin, a-fetoprotein, ferritin, copper, and ceruloplasmin; and nuclear, smooth muscle, mitoehondrial, and liver-kidney microsome autoantibodies. Moreover, 7-glutamyl transferase, albumin, and prothrombin time values obtained during the last evaluation, performed through June 1990, were also considered to evaluate liver disease. The ALT values were determined at least yearly during the follow-up, and a mean trend was drawn for a 4-year period starting from the termination of chemotherapy. A percutaneous liver biopsy specimen was obtained by using Tru-cut (Travenol Laboratories, Inc., Deerfield, Ill.) or Menghini disposable needles in 28 of 38 patients with chronic HBsAg-negative hepatitis after parents' informed consent was obtained. Twenty patients underwent liver biopsy during the first year after the cessation of chemotherapy according to a study protocol of liver histology after treatment for leukemia. Of 18 patients with another malignancy, eight underwent liver biopsy on clinical indication (i.e., chronic liver disease). Ten patients' parents did not consent to liver biopsy. Biopsy specimens were fixed in 5% buffered formalin and embedded in paraffin. Five-micromet e r sections were stained with hematoxylin and eosin, the Gomori method for reticulin, and periodic acid-Schiff reagent. The histologic diagnosis was assigned according to current criteria8: chronic active hepatitis in those with portal and periportal inflammation with erosion of the limiting plate, piecemeal necrosis, and some degree of fibrosis; chronic lobular hepatitis in those with portal tract inflammation with acute lobular features; and chronic per-
Volume 121 Number 1
HBsAg-negative hepatitis after treatment o f malignancy
Table I. Results of viral serologic studies
Table II. Blood-product units transfused to 38 patients with chronic HBsAg-negative hepatitis
Patients
Diagnosis Leukemia Other malignancy Chronic HBsAg carriers Leukemia Other malignancy HBsAg-positive, anti-HDV positive Leukemia Other malignancy Patients with chronic HBsAg-negative hepatitis Leukemia Other malignancy Patients with HBsAgnegative, HCV-positive chronic hepatitis Leukemia Other malignancy
41
No.
%
p
169/557 388/557 105/557 61/ 169 44/388 42/105
30.3 69.7 18.9 36.1 11.3 40
30/61 12/44 38/557
49.2 27.3 6.8
NS
20/169 18/388 20/38
11.8 4.6 52.6
0.004
9/169 11/388
5.3 2.8
NS
> 0.001
Leukemia (n = 20)
Average Median SD Range
14 5 18.9 0-73
Another malignancy (n = 18)
3 2 3.7 0-13
HCV infection (n = 20)
7 2 11 0-41
No HCV infection (n = 18)
11 4.5 18.4 0-73
Significance:leukemiaversusanothermalignancy,p = 0.02;HCV infection versus no HCV infection,not significant.
HDV, Hepatitisdelta virus. NS, Not significant.
sistent hepatitis when lymphocytic inflammation was restricted to portal tracts without necrosis of periportal hepatocytes. We tested HBsAg and HbcAg in the liver tissue by polyclonal antibodies (Dako Corp., Carpenteria, Calif.) and by the avidin-biotin method. Statistical analysis. Analysis of variance and covariance for statistical significance (BMDP Statistical Software, Inc. Los Angeles, Calif.) was used to compare the mean ALT trend in HCV-infected and non-HCV-infected patients. The Fisher Exact Test was used to compare group differences with respect to the diagnosis of malignancy and liver histologic findings. The Student t test was used for comparison of the number of blood product units transfused and the results of laboratory tests. Statistical comparisons were made at the 0.05 level of significance. RESULTS
Patient characteristics and results of virologic tests. Table I summarizes virologic information on 557 patients who were at least 18 months from the completion of therapy for malignancy. Two hundred fifty-two patients, 145 (86%) of 168 with a diagnosis of leukemia and 107 (27.6%) of 388 with another malignancy, had abnormal aminotransferase values during chemotherapy. Most of them had transaminase values in the normal range within a 6-month period after the cessation of chemotherapy. Thirty-eight patients (6.8%) (19 male) had chronic HBsAg-negative hepatitis; the median age at diagnosis was 4.7 (0.8 to 12.8) years; the
median follow-up from cessation of chemotherapy was 7.4 (3.9 to 9.5) years. Five of the patients did not receive any transfusion of blood products. Of 38 patients (52.6%), 20 had a positive HCV ELISA result. A significant difference was found between the leukemia and other malignancy groups (p = 0.005) but not between HCV-infected and non-HCV-infected patients, for blood product units transfused (Table II). Laboratory tests and liver disease. During the 4-year period after the cessation of chemotherapy in the patients with chronic HBsAg-negative hepatitis, there were no significant differences in mean ALT values between the HCV-infected and the non-HCV-infected patients, but there was a wide range of values in both groups. No significant difference was found between HCVinfected and non-HCV-infected patients for 3,-glutamyl transpeptidase albumin, prothrombin time, ferritin, and al-antitrypsin values. Only two HCV-infected patients, whose liver biopsy specimen revealed chronic active hepatitis, had mildly increased a-fetoprotein levels (21.13 and 27.49 ng/ml, respectively; normal range, up to 15 ng/ml). All the patients had cooper and ceruloolasmin levels in the normal range. Five HCV-infected patients had autoantibodies (1 for antinuclear autoantibody, 1 for antimicrobial autoantibody, and 3 for liver-kidney microsomal autoantibody) versus 2 HCV-negative patients (1 for antinuclear autoantibody and 1 for smooth-muscle autoantibody). These 5 patients were confirmed to have HCV infection by retesting with the HCV RIBA II test. Among 28 patients with chronic HBsAg-negative hepatitis, no significant difference was found between HCV-infected and non-HCV-infected patients for prevalence of more severe liver damage (i.e., chronic active hepatitis or cirrhosis or both; Table IIi). The presence of HBsAg and HBcAg was tested in 18 of 28 liver biopsy specimens. In four HCV-infected patients (22%), HBcAg was found in the hepatocyte nuclei. Serum from two of those four patients had transient evidence of anti-HBcAg. One patient without HCV and with high levels of serum
42
Rossetti et al.
The Journal of Pediatrics July 1992
T a b l e III. Relationship between HCV serologic studies and liver histologic diagnosis Histologic diagnosis
HCV infection (n = 20)
Cirrhosis Chronic active hepatitis Chronic lobular hepatitis Chronic persistent hepatitis Minimal changes or normal histologic findings Hemochromatosis TOTALt
1* 6* 3 4 3 0 17
No HCV infection (n = 18)
0* 1* 4 3 2 1 11
n. Total numberof patientsin the group.
*p = not significant. tTotal numberof biopsies. ferritin and iron starting 1 year after autologous bone marrow transplantation, performed for secondary leukemia, had hemochromatosis (Figure). DISCUSSION In the pediatric hemato-oncology setting, chronic liver disease is one of the major problems in long-term survivors previously treated for malignancy.2 Infection with HBV is the most frequent cause of chronic liver disease in those patients,2, 3 but the incidence of HBV infection is decreasing. Non-A, non-B virus hepatitis is now the most frequent form of posttransfusion infection.9 In our geographic area, non-A, non-B virus hepatitis represents 96% of posttransfusion hepatitis; HCV causes 87.5% of non-A, non-B hepatitis, and the prevalence of blood-product donors with anti-HCV antibody is 0.7%. l~ Hence a high prevalence of HCV infection would be expected in patients with previous malignancy who had undergone transfusion repeatedly. in this study the prevalence of chronic HCV-positive hepatitis was 3.6%. Probably because of small numbers, the prevalence of chronic HCV hepatitis was not statistically different between patients with previous leukemia and the group with another previous malignancy, as occurred in chronic HBsAg carriers. The significant difference for number of blood-product units transfused would explain the higher incidence of chronic parenteral virus infection in the leukemia group. Moreover, more severe immunodepression because of a longer treatment period and because of the leukemia itself could explain these data. No significant difference was found between HCV-infected and non-HCVinfected patients in blood-product units transfused, perhaps because chronic HBsAg-negative, HCV-negative hepatitis may be due to a virus (either HBV or HCV infection with negative serologic findings, or another virus). Two patients with no history of blood-product transfusion had HCV infection, confirming that a different route of HCV infection may be possible in our setting.4 We assume
that nondisposable material (e.g., needles for bone marrow aspirates) could have been a route of infection. The plot of the mean ALT trend in patients with chronic HBsAg-negative hepatitis is similar to the plot we previously reported for chronic HBsAg carriers who were longterm survivors of leukemia.7 In fact, in this study we observed a rapid decline of mean ALT levels during the 6-month period after the cessation of the chemotherapy, and mean ALT levels of about 100 to 150 U / L during the following years. Most patients with liver dysfunction probably caused by hepatotoxic effects of chemotherapy, and with no clinical evidence of viral hepatitis, had ALT values near the normal range within 6 months after the cessation of the chemotherapy. In this study, no liver function test indicated the likely presence of HCV infection. In particular, we confirmed for HCV-infected patients our previous observation that 3,-glutamyl transferase is an insensitive marker of the non-A, non-B carrier state. 11 Alvarez et al. 12 reported falsely positive Ortho HCV ELISA results in children with autoimmune hepatitis. In our study the specificity of the Ortho HCV ELISA in patients with autoantibodies was confirmed by the HCV RIBA II test. Even though we deal with a different group of patients (i.e, children with chronic liver disease and a previous malignancy, who are at high risk of hepatitis virus infection), we believe that a positive HCV ELISA result needs confirmation by HCV RIBA II test in patients with autoantibodies, so that proper therapy can be given. 13 We were unable to demonstrate more severe liver damage in HCV-infected patients than in the non-HCV-infected patients ones, but that hypothesis should be evaluated in a larger number of cases. Four liver specimens of non-HCV-infected patients contained HBcAg. These data confirm HBV as a cause of HBsAg-negative chronic hepatitis in patients infected during immunodepres~ion,14in particular if HBV markers were even transiently present in the serum. We conclude that HCV infection is the major cause of HBsAg-negative chronic hepatitis in patients previously treated for pediatric malignancy, and that HBV infection also can cause HBsAg-negative chronic hepatitis. Detection of HCV antigen in liver tissue might allow the diagnosis of HCV infection in patients with chronic HBsAg-negative, HCV-negative hepatitis, as described in patients with HBV infection and depressed immune function in whom HBsAg was not detected. Other non-A, non-B, non-C viruses may cause the remaining unexplained cases of chronic HBsAgnegative hepatitis. We thank Drs. A. Alberti and V. Aneloni for performing virologic tests, P. Paruzzolo, PhD, for statistical assistance, and Drs. F. Bortolotti and A. Locasciulli for reviewing the manuscript.
Volume 121 Number 1
HBsAg-negative hepatitis after treatment o f malignancy
REFERENCES 1. Zuckerman AJ. The elusive hepatitis C virus. BMJ 1989; 299:871-3. 2. Locasciulli A, Mieli Vergani G, Uderzo C, et al. Chronic liver disease in children with leukemia in long-term remission. Cancer 1983;52:1080-7. 3. Locasciulli A, Alberti, Rossetti F, et al. Acute and chronic hepatitis in childhood leukemia: a multicentric study from the Italian Pediatric Cooperative Group for Therapy of Acute Leukemia (AIL-AIEOP). Med Pediatr Oncol I985;13:203-6. 4. Rossetti F, Messina C, Pillon M, Zanesco L. Liver disease and childhood leukemia. Riv Ital Pediatr (IJP) 1991 ;17:16-22. 5. Heterington ML, Buchanan GR. Elevated serum transaminase values during therapy for acute lymphoblastic leukemia correlate with prior blood transfusion. Cancer 1988;62:1514-8. 6. Locasciulli A, Alberti A, Barbieri R, et al. Evidence of non-A, non-B hepatitis in children with acute leukemia and chronic liver disease. Am J Dis Child 1983;137:354-6. 7. Rossetti F, Zancan L, Bonato MG, et al. Delta virus and childhood leukemia. Pediatr Hematol Oncol 1991;8:23-32. 8. Bianchi L, De Groote J, Desmet U J, et al. Acute and chronic hepatitis revisited. Lancet 1977;2:914-9.
43
9. Gillon Y, Hussey A J, Howe SP, et al. Post-transfusion non-A, non-B hepatitis: significance of raised ALT and anti-HBc in blood donors. Vox Sang 1988;54:148-53. 10. Colombo M; Oldani S, Donato MF, et al. A multicenter prospective study of posttransfusion hepatitis in Milan. Hepatology 1987;7:709-12. 11. Rossetti F, Labate P, Antolini A, Zanesco L. Is gamma-glutamyl transferase a sensitive marker for detection of the non-A, non-B carrier state? [Letter]. Vox Sang 1990;58:72-3. 12. Alvarez F, Martres P, Maggiore G, Bernard O, DUssaix E. False-positive result of hepatitis C enzyme-linked immunosorbent assay in children with autoimrnune hepatitis. J PEDIATR 1991;119:75-7: 13. Davis GL. Hepatitis C virus antibody in patients with chronic autoimmune hepatitis: pitfalls in diagnosis and amplications for treatment. Mayo Clin Proc 1991;66:647-50. 14. Vergani D, Locasciulli A, Masera G, et al. Histologic evidence of hepatitis B infection with negative serology in children with acute leukemia who develop chronic liver disease. Lancet 1982;1:361-4.
alence of chronic HBsAg-negative hepatitis, to assess positivity for a marker of infection with hepatitis C virus, and to determine the severity of liver disease in this group of patients. ALT ELISA HBcAg HBsAg HBV HCV
Alanine aminotransferase Enzyme-linked immunosorbent assay HBV core antigen HBV surface antigen Hepatitis B virus Hepatitis C virus
METHODS Patients. From 1967 through June 30, 1990, a total of 557 patients were treated for malignancy at the University of Padova Department of Pediatrics, Division of Hemato-Oncology, and were at least 18 months from the completion of treatment for malignancy. Their medical records were reviewed for a history of elevated serum aminotransferase values lasting more than 12 months from the completion of
39
40
R o s s e t t i et al.
The Journal o f Pediatrics July 1992
Figure. Iron stores in periportal hepatocytes. Some portal tract macrophages are heavily laden; Perls test result is positive. (Hematoxylin-eosin stain; • chemotherapy, to identify patients with chronic HBsAgnegative hepatitis. The patients who had elevated serum aminotransferase values (normal range, 0 to 50 U / L [0.85 mmol/L]) only during the year after the chemotherapy was discontinued were not included in the study, because of the likely influence of hepatotoxic effects of chemotherapy. 4, 7 We defined chronic HBsAg-negative hepatitis as a persistent elevation of alanine aminotransferase level for more than 12 months after the cessation of chemotherapy, and with no evidence of HBsAg positivity. Virologic markers. Markers for HBV were studied at least yearly in each patient who entered the study. HBsAg, hepatitis B e antigen, anti-HBsAg, anti-hepatitis B e antigen and anti-hepatitis B core antigen antibodies were tested by radioimmunoassay or by enzymatic immunoassay (Abbott Laboratories, Diagnostic Division, Latina, Italy, or Sorin Biomedica SPA, Saluggia, italy). Anti-HCV antibody was measured by the Ortho HCV enzyme-linked immunosorbent assay (Ortho Diagnostic Systems, Milan, Italy) in patients with chronic HBsAg-negative hepatitis. In accordance with instructions provided by the manufacturer, the test was done in microwell plates by an automated procedure. Each plate included one blank and three negative and two positive control specimens. The cutoff value was calculated by adding 0.400 to the mean value for negative control specimens. The specimens were considered to be definitely reactive if adsorbance values above the cutoff point were confirmed on retesti~i~Six sera from patients with autoantibodies were retested with a recombinant immunoblot test (HCV RIBA II test, Chiron Corp., Emeryville, Calif.) specific for HCV antibodies. Assessment of hepatic disease. After the completion of
treatment for malignancy, the following tests were performed, at least once, to rule out other causes of chronic liver disease in the patients with chronic HBsAg-negative hepatitis: serum cq-antitrypsin, a-fetoprotein, ferritin, copper, and ceruloplasmin; and nuclear, smooth muscle, mitoehondrial, and liver-kidney microsome autoantibodies. Moreover, 7-glutamyl transferase, albumin, and prothrombin time values obtained during the last evaluation, performed through June 1990, were also considered to evaluate liver disease. The ALT values were determined at least yearly during the follow-up, and a mean trend was drawn for a 4-year period starting from the termination of chemotherapy. A percutaneous liver biopsy specimen was obtained by using Tru-cut (Travenol Laboratories, Inc., Deerfield, Ill.) or Menghini disposable needles in 28 of 38 patients with chronic HBsAg-negative hepatitis after parents' informed consent was obtained. Twenty patients underwent liver biopsy during the first year after the cessation of chemotherapy according to a study protocol of liver histology after treatment for leukemia. Of 18 patients with another malignancy, eight underwent liver biopsy on clinical indication (i.e., chronic liver disease). Ten patients' parents did not consent to liver biopsy. Biopsy specimens were fixed in 5% buffered formalin and embedded in paraffin. Five-micromet e r sections were stained with hematoxylin and eosin, the Gomori method for reticulin, and periodic acid-Schiff reagent. The histologic diagnosis was assigned according to current criteria8: chronic active hepatitis in those with portal and periportal inflammation with erosion of the limiting plate, piecemeal necrosis, and some degree of fibrosis; chronic lobular hepatitis in those with portal tract inflammation with acute lobular features; and chronic per-
Volume 121 Number 1
HBsAg-negative hepatitis after treatment o f malignancy
Table I. Results of viral serologic studies
Table II. Blood-product units transfused to 38 patients with chronic HBsAg-negative hepatitis
Patients
Diagnosis Leukemia Other malignancy Chronic HBsAg carriers Leukemia Other malignancy HBsAg-positive, anti-HDV positive Leukemia Other malignancy Patients with chronic HBsAg-negative hepatitis Leukemia Other malignancy Patients with HBsAgnegative, HCV-positive chronic hepatitis Leukemia Other malignancy
41
No.
%
p
169/557 388/557 105/557 61/ 169 44/388 42/105
30.3 69.7 18.9 36.1 11.3 40
30/61 12/44 38/557
49.2 27.3 6.8
NS
20/169 18/388 20/38
11.8 4.6 52.6
0.004
9/169 11/388
5.3 2.8
NS
> 0.001
Leukemia (n = 20)
Average Median SD Range
14 5 18.9 0-73
Another malignancy (n = 18)
3 2 3.7 0-13
HCV infection (n = 20)
7 2 11 0-41
No HCV infection (n = 18)
11 4.5 18.4 0-73
Significance:leukemiaversusanothermalignancy,p = 0.02;HCV infection versus no HCV infection,not significant.
HDV, Hepatitisdelta virus. NS, Not significant.
sistent hepatitis when lymphocytic inflammation was restricted to portal tracts without necrosis of periportal hepatocytes. We tested HBsAg and HbcAg in the liver tissue by polyclonal antibodies (Dako Corp., Carpenteria, Calif.) and by the avidin-biotin method. Statistical analysis. Analysis of variance and covariance for statistical significance (BMDP Statistical Software, Inc. Los Angeles, Calif.) was used to compare the mean ALT trend in HCV-infected and non-HCV-infected patients. The Fisher Exact Test was used to compare group differences with respect to the diagnosis of malignancy and liver histologic findings. The Student t test was used for comparison of the number of blood product units transfused and the results of laboratory tests. Statistical comparisons were made at the 0.05 level of significance. RESULTS
Patient characteristics and results of virologic tests. Table I summarizes virologic information on 557 patients who were at least 18 months from the completion of therapy for malignancy. Two hundred fifty-two patients, 145 (86%) of 168 with a diagnosis of leukemia and 107 (27.6%) of 388 with another malignancy, had abnormal aminotransferase values during chemotherapy. Most of them had transaminase values in the normal range within a 6-month period after the cessation of chemotherapy. Thirty-eight patients (6.8%) (19 male) had chronic HBsAg-negative hepatitis; the median age at diagnosis was 4.7 (0.8 to 12.8) years; the
median follow-up from cessation of chemotherapy was 7.4 (3.9 to 9.5) years. Five of the patients did not receive any transfusion of blood products. Of 38 patients (52.6%), 20 had a positive HCV ELISA result. A significant difference was found between the leukemia and other malignancy groups (p = 0.005) but not between HCV-infected and non-HCV-infected patients, for blood product units transfused (Table II). Laboratory tests and liver disease. During the 4-year period after the cessation of chemotherapy in the patients with chronic HBsAg-negative hepatitis, there were no significant differences in mean ALT values between the HCV-infected and the non-HCV-infected patients, but there was a wide range of values in both groups. No significant difference was found between HCVinfected and non-HCV-infected patients for 3,-glutamyl transpeptidase albumin, prothrombin time, ferritin, and al-antitrypsin values. Only two HCV-infected patients, whose liver biopsy specimen revealed chronic active hepatitis, had mildly increased a-fetoprotein levels (21.13 and 27.49 ng/ml, respectively; normal range, up to 15 ng/ml). All the patients had cooper and ceruloolasmin levels in the normal range. Five HCV-infected patients had autoantibodies (1 for antinuclear autoantibody, 1 for antimicrobial autoantibody, and 3 for liver-kidney microsomal autoantibody) versus 2 HCV-negative patients (1 for antinuclear autoantibody and 1 for smooth-muscle autoantibody). These 5 patients were confirmed to have HCV infection by retesting with the HCV RIBA II test. Among 28 patients with chronic HBsAg-negative hepatitis, no significant difference was found between HCV-infected and non-HCV-infected patients for prevalence of more severe liver damage (i.e., chronic active hepatitis or cirrhosis or both; Table IIi). The presence of HBsAg and HBcAg was tested in 18 of 28 liver biopsy specimens. In four HCV-infected patients (22%), HBcAg was found in the hepatocyte nuclei. Serum from two of those four patients had transient evidence of anti-HBcAg. One patient without HCV and with high levels of serum
42
Rossetti et al.
The Journal of Pediatrics July 1992
T a b l e III. Relationship between HCV serologic studies and liver histologic diagnosis Histologic diagnosis
HCV infection (n = 20)
Cirrhosis Chronic active hepatitis Chronic lobular hepatitis Chronic persistent hepatitis Minimal changes or normal histologic findings Hemochromatosis TOTALt
1* 6* 3 4 3 0 17
No HCV infection (n = 18)
0* 1* 4 3 2 1 11
n. Total numberof patientsin the group.
*p = not significant. tTotal numberof biopsies. ferritin and iron starting 1 year after autologous bone marrow transplantation, performed for secondary leukemia, had hemochromatosis (Figure). DISCUSSION In the pediatric hemato-oncology setting, chronic liver disease is one of the major problems in long-term survivors previously treated for malignancy.2 Infection with HBV is the most frequent cause of chronic liver disease in those patients,2, 3 but the incidence of HBV infection is decreasing. Non-A, non-B virus hepatitis is now the most frequent form of posttransfusion infection.9 In our geographic area, non-A, non-B virus hepatitis represents 96% of posttransfusion hepatitis; HCV causes 87.5% of non-A, non-B hepatitis, and the prevalence of blood-product donors with anti-HCV antibody is 0.7%. l~ Hence a high prevalence of HCV infection would be expected in patients with previous malignancy who had undergone transfusion repeatedly. in this study the prevalence of chronic HCV-positive hepatitis was 3.6%. Probably because of small numbers, the prevalence of chronic HCV hepatitis was not statistically different between patients with previous leukemia and the group with another previous malignancy, as occurred in chronic HBsAg carriers. The significant difference for number of blood-product units transfused would explain the higher incidence of chronic parenteral virus infection in the leukemia group. Moreover, more severe immunodepression because of a longer treatment period and because of the leukemia itself could explain these data. No significant difference was found between HCV-infected and non-HCVinfected patients in blood-product units transfused, perhaps because chronic HBsAg-negative, HCV-negative hepatitis may be due to a virus (either HBV or HCV infection with negative serologic findings, or another virus). Two patients with no history of blood-product transfusion had HCV infection, confirming that a different route of HCV infection may be possible in our setting.4 We assume
that nondisposable material (e.g., needles for bone marrow aspirates) could have been a route of infection. The plot of the mean ALT trend in patients with chronic HBsAg-negative hepatitis is similar to the plot we previously reported for chronic HBsAg carriers who were longterm survivors of leukemia.7 In fact, in this study we observed a rapid decline of mean ALT levels during the 6-month period after the cessation of the chemotherapy, and mean ALT levels of about 100 to 150 U / L during the following years. Most patients with liver dysfunction probably caused by hepatotoxic effects of chemotherapy, and with no clinical evidence of viral hepatitis, had ALT values near the normal range within 6 months after the cessation of the chemotherapy. In this study, no liver function test indicated the likely presence of HCV infection. In particular, we confirmed for HCV-infected patients our previous observation that 3,-glutamyl transferase is an insensitive marker of the non-A, non-B carrier state. 11 Alvarez et al. 12 reported falsely positive Ortho HCV ELISA results in children with autoimmune hepatitis. In our study the specificity of the Ortho HCV ELISA in patients with autoantibodies was confirmed by the HCV RIBA II test. Even though we deal with a different group of patients (i.e, children with chronic liver disease and a previous malignancy, who are at high risk of hepatitis virus infection), we believe that a positive HCV ELISA result needs confirmation by HCV RIBA II test in patients with autoantibodies, so that proper therapy can be given. 13 We were unable to demonstrate more severe liver damage in HCV-infected patients than in the non-HCV-infected patients ones, but that hypothesis should be evaluated in a larger number of cases. Four liver specimens of non-HCV-infected patients contained HBcAg. These data confirm HBV as a cause of HBsAg-negative chronic hepatitis in patients infected during immunodepres~ion,14in particular if HBV markers were even transiently present in the serum. We conclude that HCV infection is the major cause of HBsAg-negative chronic hepatitis in patients previously treated for pediatric malignancy, and that HBV infection also can cause HBsAg-negative chronic hepatitis. Detection of HCV antigen in liver tissue might allow the diagnosis of HCV infection in patients with chronic HBsAg-negative, HCV-negative hepatitis, as described in patients with HBV infection and depressed immune function in whom HBsAg was not detected. Other non-A, non-B, non-C viruses may cause the remaining unexplained cases of chronic HBsAgnegative hepatitis. We thank Drs. A. Alberti and V. Aneloni for performing virologic tests, P. Paruzzolo, PhD, for statistical assistance, and Drs. F. Bortolotti and A. Locasciulli for reviewing the manuscript.
Volume 121 Number 1
HBsAg-negative hepatitis after treatment o f malignancy
REFERENCES 1. Zuckerman AJ. The elusive hepatitis C virus. BMJ 1989; 299:871-3. 2. Locasciulli A, Mieli Vergani G, Uderzo C, et al. Chronic liver disease in children with leukemia in long-term remission. Cancer 1983;52:1080-7. 3. Locasciulli A, Alberti, Rossetti F, et al. Acute and chronic hepatitis in childhood leukemia: a multicentric study from the Italian Pediatric Cooperative Group for Therapy of Acute Leukemia (AIL-AIEOP). Med Pediatr Oncol I985;13:203-6. 4. Rossetti F, Messina C, Pillon M, Zanesco L. Liver disease and childhood leukemia. Riv Ital Pediatr (IJP) 1991 ;17:16-22. 5. Heterington ML, Buchanan GR. Elevated serum transaminase values during therapy for acute lymphoblastic leukemia correlate with prior blood transfusion. Cancer 1988;62:1514-8. 6. Locasciulli A, Alberti A, Barbieri R, et al. Evidence of non-A, non-B hepatitis in children with acute leukemia and chronic liver disease. Am J Dis Child 1983;137:354-6. 7. Rossetti F, Zancan L, Bonato MG, et al. Delta virus and childhood leukemia. Pediatr Hematol Oncol 1991;8:23-32. 8. Bianchi L, De Groote J, Desmet U J, et al. Acute and chronic hepatitis revisited. Lancet 1977;2:914-9.
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9. Gillon Y, Hussey A J, Howe SP, et al. Post-transfusion non-A, non-B hepatitis: significance of raised ALT and anti-HBc in blood donors. Vox Sang 1988;54:148-53. 10. Colombo M; Oldani S, Donato MF, et al. A multicenter prospective study of posttransfusion hepatitis in Milan. Hepatology 1987;7:709-12. 11. Rossetti F, Labate P, Antolini A, Zanesco L. Is gamma-glutamyl transferase a sensitive marker for detection of the non-A, non-B carrier state? [Letter]. Vox Sang 1990;58:72-3. 12. Alvarez F, Martres P, Maggiore G, Bernard O, DUssaix E. False-positive result of hepatitis C enzyme-linked immunosorbent assay in children with autoimrnune hepatitis. J PEDIATR 1991;119:75-7: 13. Davis GL. Hepatitis C virus antibody in patients with chronic autoimmune hepatitis: pitfalls in diagnosis and amplications for treatment. Mayo Clin Proc 1991;66:647-50. 14. Vergani D, Locasciulli A, Masera G, et al. Histologic evidence of hepatitis B infection with negative serology in children with acute leukemia who develop chronic liver disease. Lancet 1982;1:361-4.
