Portmann B, O'Grady JG, Williams R: Disease recurrence following orthotopic liver .... Govindarajan S, Edwards VM, Stuart ML, Operskalski EA,. Mosely JW, the ...
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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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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Steatoviral and Fibroviral Hepatitis B
1301
AJP June 1992, Vol. 140, No. 6
markers but no hepatitis developed during the period of observation. With progression, an increase in the disarray of the hepatocytes, variation in the size and shape of hepatocytes, and variation in their staining occurred; in some cells, the cytoplasm was more acidophilic, others were paler stained and more swollen or ballooned. The progression of the liver lesion occurred along one of two pathways. In six of the nine cases with recurrent HBV hepatitis (Table 3), fat droplets were noted in many of the liver cells (steatoviral hepatitis B) (Figure 1f, g). The steatosis was either macrovesicular or microvesicular (Figure 1g, h). Small fat droplets were frequently so numerous that the entire cytoplasm was expanded and had a bubbly appearance. Mallory bodies were not found. The appearance of these biopsy specimens was striking, with lipid-filled hepatocytes, ballooned cells, typical groundglass cells, pale staining ground- glass cells, and large swollen cells having pale-staining, bubbly, glassy, or clear cytoplasm. Necro-inflammatory changes with mononuclear cell aggregates and scattered acidophilic bodies could easily be found. Canalicular cholestasis in zone 3 areas was variable. Chronic active hepatitis with aggressive piece-meal necrosis and fibrosis developed. Once steato-hepatitic features appeared, they tended to become widespread and eventually almost all lobules showed these features. An exception was the presence of small foci of essentially normal liver cells that appeared resistant to the changes described. Mitotic figures, biand trinucleated cells were infrequent except in the resistant normal-appearing cells. Subsequently, cellular necrosis, collapse, and fibrosis were found (Figure 1 i). In the preterminal stage, the swollen fatty and clear cytoplasm of hepatocytes became bile-stained. By electron microscopy, the balloon cells showed markedly dilated endoplasmic reticulum containing flocculent material, ground-glass cells had a typical appearance with strands of filamentous viral protein (HBsAg) in the endoplasmic reticulum, and the cells with bubbly cytoplasm were filled with small fat droplets. In the typical cell of steatoviral hepatitis B, a mix of these findings was seen. The endoplasmic reticulum was widely distended (as in balloon cells) but filled or partially filled with HBsAg particles in a manner not noted before, and lipid droplets were also seen (Figure 2). Dane particles were found in the dilated sacs of the endoplasmic reticulum with the HBsAg filaments. The sanded nuclei of HBcAg-positive cells showed HBV-core particles admixed with the chromatin material. The liver cells with the eosinophilic nuclear inclusions showed massive numbers of core particles,
more than a million
in some nuclei (Figure 3). With further progression of the disease, necrosis and inflammation increased, liver lobules were reduced in size and the surviving cells were extremely swollen. There was intralobular and pericellular fibrosis. Cholestasis was common and was centrilobular and canalicular in distribution. In later stages, periportal and intralobular inflammation, piecemeal necrosis, and fibrosis were more prominent; the liver cells were even more swollen, and bile-stained granules, often in large numbers, appeared in their cytoplasm. When the biopsy showed a predominance of swollen bile-staisned cells, the prognosis was unfavorable. In the second variant (fibroviral hepatitis B) of recurrent hepatitis (four of the nine cases, Table 3) the route to progression was that of increasing disarray of liver cells, increase in ground glass cells, and increasing pericellular and intralobular fibrosis but without the appearance of fatty, clear, and ballooned cells. In this pathologic variant, inflammatory changes and overt cellular necrosis with Councilman-like bodies again tended to be mild even in advanced stages of the disease, but progression of the disease was the rule. By electron microscopy, nuclear changes were characterized by extreme positivity for core particles whereas the cytoplasm of the ground-glass cells had the typical appearance of filamentous strands and spherical HBsAg particles in the cisternae of the endoplasmic reticulum. The fibrosis was out of proportion to the necro-inflammatory changes seen.
Cholestasis is another important facet of the histopathology and is seen under two main circumstances. In the transition from the viral overload state to true hepatitis when necro-inflammatory changes appear, centrilobular canalicular cholestasis with bile plugs are common findings. However, the cholestasis does not persist and becomes less obvious or disappears as other changes ensue. Cholestasis returns in different form as a significant manifestation late in the disease when entire hepatocytes become swollen and bile stained. By electron microscopy, these cells show multifocal cytoplasmic degeneration and bile necrosis. Six of the patients with recurrent hepatitis B died, three with severe chronic hepatitis and liver failure, one from a myocardial infarct, one with sepsis, and one with disseminated immunoblastic lymphoma. The three patients who died with severe hepatitis had steatoviral hepatitis B. One of the six patients in the nonrecurrent group died 3 years post-transplantation of cerebral hemorrhage and hypertension.
Figure 2. Electron micrograph of typical clear cellfound in recurrentsteato-viral hepatitisB. Note the rough endoplasmic reticulum is widely dilated and contains an abundance of HBsAg particles. Note also the small lipid droplets in the adjacent cytoplasm. Uranyl acetate and lead citrate stain, x 45,500.
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irC.._c 5;
Steatoviral and Fibroviral Hepatitis B 1303 AJP June 1992, Vol. 140, No. 6
Types of HBV Response Post-Transplant Donor liver
No Recurrence
Recurrence f viral markers
I
I
No Progression
Massive viral burden
necro-inflammatory disease with cholestasis fibrosis
Fibroviral Hepatitis B
_A
steatosis
Steatoviral Hepatitis B
Figure 4. Schematic representation of the sequence ofprogression seen in recurrent hepatitis B, post-liver transplantation.
Time-Sequence of Pathologic Findings Progression of the disease was the rule although several of the patients had their disease arrested or reversed for long periods by PGE treatment. The relationship of the histopathologic and immunopathologic changes in relationship to each other and the time-course of the disease are important considerations. Five patients remained free of hepatitis B disease clinically and pathologically; serum and tissue markers have remained negative to date (mean follow-up, 826 days; range, 125-1095 days). In the remaining 11 patients with hepatitis B, the interval between transplantation and the first evidence of positive markers in the liver postoperatively averaged 118 days (range, 12-307 days). An exception is the patient receiving two transplants (OLT 166). In this patient, positive markers recurred 80 days after the first transplant and after only 12 days in the second graft. Other reports also stress the accelerated pace of recurrent disease with
multiple retransplantations.311 19 The tissue markers for HBsAg and HBcAg invariably started as focal slight positively scattered hepatocytes (+), but progressed in every instance to marked severity usually involving greater than 75% to 95% of hepatocytes (+ + + +). Frequently the progression from minimal to marked positivity was explosive, occurring over a matter of weeks, and it commonly preceded the development of necro-inflammatory or other changes, often by months. Usually the initial positivity of markers occurred while the liver appeared histologically normal or showed only slight disarray of hepatocytes. The appearance of histologic evidence of lobular or periportal hepatitis occurred at an average of 156 days (range, 1 15-307 days) post-transplantation. The relationship to PGE treatment is also important since this form of management is unique to this series of patients. The results are shown in Tables 3 and 4. PGE appeared to have a modulating effect on the posttransplant HBV changes in some but not all instances
Figure 3. Electron micrograph of bepatocyte nucleus in recurrent fibro-viral hepatitis B. Only part of one nucleus is shown but it is representative of the massive viral burden ofcore particles in nuclei The small circular particles are HBV core particles. Chromatin is scanty and widely scattered Uranyl acetate stain, x 18,360. Note: Based on this and similar electron micrographs, an estimate was made of the number of core particles in this patient's liver (OLT 080). The following assumptions were made: The nucleus is a sphere. There are a normal number of cells in the liver. There are 250 billion hepatocytes in the adult liver (Arias IM, et al;
