American Journal of PathologD,, Vol. 140, No. 6, June 1992 Copyright C) American Association of Pathologists

Post-transplant Recurrent Hepatitis B Viral Liver Disease Viral-burden, Steatoviral, and Fibroviral Hepatitis B

M. J. Phillips,*l1 R. Cameron,*l1 M. A. Flowers,11 L. M. Blendis,t"l P. D. Greig,tll 1. Wanless,*lI M. Sherman,til R. Superina,til B. Langer,t4 and G. A. Levytil Medicine,t and Surgery,* The Hospitalfor Sick Children, Th-e Toronto

From the Departments of Pathology, *

Hospital, Toronto, and the University of Toronto,"1 Toronto, Ontario, Canada

time of hepatic resection, sufficient virus persists in extrahepatic sites to lead to reinfection of the allograft in more than half of the patients.1" Of the 192 patients undergoing liver transplantation from September 1985 to March 1991 at this center, 26 had HBV-associated liver disease. The clinical findings, laboratory results, and management of these patients are reported elsewhere,45 so only selected aspects of these findings are discussed here. There have been a number of publications on recurrence of hepatitis B post-transplantation,1-11 including an article describing distinctive histopathologic features (cholestasis and fibrosis).12 This article describes unique characteristics of the liver pathology found in the 16 hepatitis B (HBV) positive patients in whom there is long-term follow-up (1 00-1234 days). Two characteristics of recurrent HBV liver disease are emphasized: the massive viral load that is found, and the tendency to progression along one of two paths - hepatitis with progressive fibrosis, and hepatitis with small or large droplet steatosis.

Recurrence of hepatitis is a well-documented complication of hepatitis B liver disease, post-transplantation. It is well established also that the earliest hepatocellular change is the appearance of hepatitis B viral (HBV) markers and that the disease is rapidly progressive. In this article on 17 liver transplants in 16 HBV positive patients with long-term follow-ups (100-1234 days), the distinctive pathologic features of this disease are emphasized: the extreme viral load, the steatosis, and/or fibrosis. An attempt to quantitate the magnitude of the viral burden was made and the result was a staggering figure. In one patient, an estimated 10j8 HBV core particles were present in the liver. One of two patterns of progression were noted. In four patients in addition to the massive nuclear hepatitis B core antigen (HBcAg) and cytoplasmic hepatitis B surface antigen (HBsAg) positivity, superimposed hepatitic changes led to diffuse hepatic fibrosis (fibroviral hepatitis B); and in another six patients, extraordinar hepatocellular viral marker positivity and steatosis were the hallmarks (steatoviral hepatitis B). Steatosis is not usually considered a feature of HBV liver pathology. These results suggest that more than one type ofposttransfusion recurrent hepatitis B liver disease exists pathologically. (Am JPathol 1992, 140:1295-1308)

The liver pathology of the 16 patients in the study form the basis of this study; the recipient livers and 45 posttransplant liver biopsy specimens were examined from these patients (Table 1). One of the patients had two liver transplants. Routine pathologic study included an evaluation of formalin-fixed, paraffin-embedded liver tissue sections stained by hematoxylin and eosin (H&E), Masson's trichrome, periodic acid Schiff (PAS), PAS with diastase, orcein, and reticulin stains.13 All samples were examined by the peroxidase antiperoxidase method (PAP)14 using antisera against HB-

Orthotopic liver transplantation (OLT) is rapidly becoming the treatment of choice for many forms of end-stage chronic liver disease but its role in the management of chronic viral hepatitis B (HBV) infection remains controversial. Despite removal of the bulk of the viral load at the

Supported by a grant in aid from the Medical Research Council of Canada (M.J.P., MRC 0785). Accepted for publication December 30, 1991. Address reprint requests to Dr. M. J. Phillips, Department of Pathology, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada M5G 1 X8.

Materials and Methods

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1296

Phillips et al

AJPJune 1992, Vol. 140, No. 6

disease and were compared with the findings reported in

Table 1. Summary ofResults 17 Liver Transplants in 16 Hepatitis B Patients 5/17 12/17 3/17 9/17

6/16* *

nontransplant hepatitis B liver disease.17

No recurrence Initial postoperative biopsies were normal Positive HBV markers was the first biopsy evidence of recurrence Recurrence of HBV tissue markers only Recurrence of HBV markers and histological hepatitis 6/9 steatoviral hepatitis B 3/9 fibroviral hepatitis B Died; 3/6 were unrelated to HBV

Clinical Management

There were 16 patients, one had two transplants.

sAg (Calbiochem, La Jolla, CA) and HBcAg (Dakopatts, Glostrup, Denmark). Sections were incubated in 0.05% pepsin (Sigma, St Louis, MO) in 0.01% HCI at 37°C for 30 minutes, then blocked with methanol and hydrogen peroxide mixture for 30 minutes. After treatment with normal swine serum (1/10 dilution) (Dakopatts, Glostrup, Denmark), sections were incubated with antisera against HBsAg (1/200 dilution) and HBcAg (1/100 dilution) for 1 hour, then they were further incubated with swine antirabbit immunoglobulins (1/50 dilution) for 30 minutes followed by rabbit peroxidase anti-peroxidase complex (1/ 50 dilution) for 30 minutes (both from Dakopatts, Glostrup, Denmark). The peroxidase reaction was visualized using 3, 3'-diaminobenzidine (DAB) (Sigma, St Louis, MO) in TRIS buffer (pH 7.6) containing hydrogen peroxide. The scoring system used for assessing the number of (HBsAg, HBsAg) positive cells was as follows: - (0), + (1-25%), + + (25-50%), + + + (50-75%), + + + + (75-100%). Electron microscopy was carried out on all the cases. The electron microscopy tissue processing procedures used were those described by Oda et al15 and electron microscopy staining methods described by Sato.'6 Electron micrographs were examined from all stages of the

The patients were closely monitored clinically and by laboratory studies. Tests results on the serum of liver donors for HBsAg and HIV before transplant were negative. The serum of the liver recipients was tested for HBsAg and HBV-DNA before transplantation; all were HBsAgpositive and all but four were HBV-DNA-negative (Tables 2, 3). All the tests that were done for hepatitis D virus (HDV) were negative; all patients were hepatitis C virus (HCV) negative. Biochemical evidence of recurrent HBV infection was detected in the serum from 45 to 330 days. The correlation between the degree of elevation of serum transaminases, the prothrombin time and the histologic grade of hepatitis was variable. Elevation of serum transaminases did not consistently precede detection of positive immunostaining for viral antigens. All patients received standard immunosuppressive therapy consisting of corticosteroids and cyclosporin A. Treatment with prostaglandin E (PGE2, by continuous IV infusion) in eight patients (OLT# 038, 041, 057, 126, 139, 140, 150, 174) and oral PGE1, in two others (OLT# 080,179) was initiated from 130 to 330 days postoperatively, with an average 60-day interval between first detection of recurrent disease and institution of PGE therapy. The dates at which PGE treatment began in relation to the stage of the disease pathologically are shown in Tables 3 and 4. Four patients (OLT# 041, 126,150, 174) had one episode of acute rejection, two (OLT# 038,139) had two episodes, and three (OLT# 080, 149, 179) had three episodes; all were in the early postoperative period. The criteria proposed by Snover et al18 were used in the diagnosis and

Table 2. No Recurrence Group HBV disease

Recipient liver Tissue

Serum

*OLT no.

HBsAg

009

+

HBV DNA -

Histology

Cirrhosis

Donor liver

HBsAg

Markers HBcAg

Histology

Patient status (days)

+

+

Normal

-

-

Normal

Died, hypertension cerebral hemorrhage (805) Alive and well (1095)

+++

-

Normal

Alive and well

(cyto) 055

+

NT

057

+

-

Acute Fulminant Hepatitis CAH

(cyto)

(1085)

067

+

-

Cirrhosis

+

-

Normal

Alive and well

163

+

NT

Cirrhosis

(cyto) + (cyto)

-

Normal

Alive and well

(1022) (125)

OLT No., Patient orthotopic liver transplant number; NT, not tested; Cyto, cytoplasmic; CAH, chronic active hepatitis.

Steatoviral and Fibroviral Hepatitis B

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AJPJune 1992, Vol. 140, No. 6

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