Fibrosing cholestatic hepatitis in a transplant recipient with hepatitis B virus precore mutant

GASTROENTEROLOGY

1993;105:901-904

CASE REPORTS Fibrosing Cholestatic Hepatitis in a Transplant Hepatitis B Virus Precore Mutant JANE W. S. FANG,* FRANK Y. T. TUNG,* GARY L. DAVIS,” DAVID H. VAN THIEL,’ and JOHNSON Y. N. LAU*

DAVID

Recipient With

J. DOLSON,*

*Section of Hepatobiliary Diseases, Divisron of Gastroenterology, Hepatology, and Nutrition, and *Department of Pathology and Laboratory Medicine, University of Florida College of Medicine, Gainesville, Florida; and “Oklahoma Transplantation Institute at Baptist Medical Center of Oklahoma, Oklahoma City, Oklahoma

A patient with hepatitis B virus (HBV) precore mutant (seropositive for hepatitis B surface antigen [HBsAgl, anti-hepatitis B e antigen (HBeAgl, and HBV DNA) who underwent orthotopic liver transplantation for endstage liver disease is described. Sequencing of the HBV precore region of the pretransplant serum sample confirmed the presence of the precore stop-codon mutant (G + A mutation in codon 1896) only. The patient received HBV immunoglobulin prophylaxis for 6 months but HBV recurred thereafter with a mild hepatitic flare, and he remained seropositive for HBsAg, anti-HBe, and HBV DNA. The initial hepatitic illness resolved in 3 months. The patient remained well for another 16 months before presenting with fibrosing cholestatic hepatitis (FCH). During his entire initial hepatitic flare, quiescent period, and final FCH phase, he remained seropositive for HBsAg, anti-HBe, and HBV DNA. Moreover, sequencing of the serum HBV DNA in final FCHphase showed the presence of the identical HBV precore mutant. Immunohistochemical staining showed extensive expression of HBsAg/pre-S, , preS,, and hepatitis B core antigen, but HBeAg was scarcely detectable. This case illustrates that (1) recurrente of HBV precore mutant infection can occur in liver; (2) it can give rise to FCH; and (3) hepatic accumulation of HBeAg is not essential for the development of FCH.

C

hronic

hepatitis

B virus (HBV)

infection

is poten-

tially one of the most common indications for liver transplantation worldwide. Unfortunately, recurrente rate of HBV infection in the allograft liver is extremely high and is associated with increased morbidity and mortality after transplantation.‘*’ A proportion of the patients with HBV recurrence develop a peculiar syndrome called fibrosing cholestatic hepatitis (FCH), which is characterized clinically by rapidly progressive hepatic failure and a resultant very high mortality rate within 4-6 weeks of clinical onset. Histologically, it is characterized by a pattern of extensive

serpiginous cholestasis, patocytes

periportal

fibrosis,

prominent

canalicular

HBV antigen

but, paradoxically,

and cellular

expression

a mild

mixed

in he-

inflamma-

torv, cel1 infiltrate.2-5 The clinical and histological pattern, as wel1 as the high leve1 of HBV antigen expression, condition

suggested

that the liver cel1 damage

may result

directly

fect of the overexpression In the cases reported gens (hepatitis Sa, hepatitis antigen

so far, al1 detectable

HBV

(HBeAg

liver of these patients.

ef-

at high

that

is actively lacking

FCH after orthotopic

report, HBeAg

B e

levels

the

in

wild-type

replicating

In the present

mutant

HBV antipre-S, , pre-

and hepatitis

found

suggesting

positive)

HBV precore

[HBsAg],

[HBcAg],

h ave been

[HBeAg])

developed

antigen

B core antigen

hepatocytes,5,6

in this

the cytopathic

of HBV antigens.2’4

B surface

most

with

from

in the a patient

expression

liver transplantation.

The histological picture of this case was similar to that reported for other cases of FCH except that only occasional

hepatocytes

pathogenetic

expressed

implications

HBeAg.

The clinical

and

of this case are discussed.

Case Report A 41-year-old

white man presented in December 1989 with esophageal variceal hemorrhage that was subsequently controlled by repeated sclerotherapy. He was found to have both clinical and biochemical evidente of cirrhosis including a smal1 liver span, hypersplenism, and a low serum albumin leve1 (2.7 g/‘dL; normal, 3-5 g/dL). He had normal (AST),

aminotransferase 35 IU/L (normal,

levels: aspartate aminotransferase 0-40 IU/L); and alanine amino-

(ALT), 24 IU/L (normal, 0-40 IU/L). A hepatitis B serologie assessment showed that the patient was positive for HBsAg and antibody to HBeAg (anti-HBe). He was

transferase

Abbreviations used in this paper: FCH, flbrosing cholestatic hepatitis; HBIG, hepatitis B virus immunoglobulin (prophylaxis). 6211993 by the American Gastroenterological Association

0016-5085/93/$3.00

902

FANG ET AL.

negative

for both hepatitis

nodeficiency

virus

performed quent

GASTROENTEROLOGY Vol. 105, No. 3

D (delta) virus and human

markers.

at the time of initial

study of the stored

plantation DNA.

A HBV

showed

that

Sequencing

fied fragment mutation

serum

but a subse-

after successful

(TAG)

of the wild-type

form,

indicating

that

region.

codon

type of HBV present

in his serum that

could

creat-

received

for end-stage

elective

orthotopic

erative course was uneventful

Sequencing

in the patient’s

ence

present

of the identical obtained

type

characteristic

features

Profound

liver transplan-

noted.

with features ballooning

and a minima1

inflammatory bile stasis

Immunohistochemical

(al1 4+)

in most

with

in the

with no signal specimens

showed

increased

pericellu-

suggestive

of cirrho-

with

ground

showed

nuclear

glass

was noted

in hepatocytes

HBsAg/pre-S,/pre-S,,

hepatocytes

the pres-

found

infiltrate

staining

for

region of the

showed

mutant

biopsy

of FCH including

fibrosis

of cytoplasmic

HBV

Liver

hepatocyte

position

serum

liver transplantation,

HBV.

1). Prominent

test results

immunodeficiency

of the precore

precore

before

for the wild

(Figure to

serologie

HBV DNA

changes

him

Repeated

D virus, and human

virus were negative.

lar and bridging

liver transplanta-

DNA.

hepatitis

sis.

predisposed

liver disease in April,

HBV

in the wave-

was identified.

The patient

and

serum

a point

was the dominant

before have

HBe,

anti-HCV,

There was no

observed

mutant

tion.

HBV intection

for HBV

in the HBV DNA,

that the precore

NO risk factor

positive

showed

in the precore TGG

liver trans-

chain reaction-ampli-

region

1896 existed

signal

tation

presentation,

of the polymerase

at position

inga stop codon

assay was not

he was weakly

of the precore

immu-

DNA

was also

extensive

de-

and HBcAg HBcAg

in only

1990. The postop-

apart from mild hepatic

injury

proven to have been caused by graft ischemia and the development of a focal portal venous endothelialitis consistent with an acute allograft postliver given

rejection

transplant

a 6-month

biopsy

course

episode

shown

specimens.

The

in the initial patient

of HBV immunoglobulin

laxis (HBIG,

10,000 units monthly

treated

with

a combination

prevent

allograft

for 6 months)

of FK506

rejection

was

prophyand was

and prednisone

at a maintenance

to

dosage of 12 mg

orally twice daily and 10 mg orally every day, respectively. He remained

seropositive

and anti-HBe

during

munoprophylaxis. phylaxis,

Shortly

he was found

anti-HBe,

for antibody

to HBsAg (anti-HBs)

the time he was receiving after the cessation

to be seropositive

and HBV DNA.

ALT, 136 IU/L)

tion with an elevated

total bilirubin

mg/dL

(normal,

the HBIG.

opportunistic staining

infections

cytoplasmic core

a mild lobular were seen.

(HBcAg

HBV DNA

cytoplasm

sections

3f)

disease

re-

or other

Immunohistochemical

absente

the presence 3f)

of detectable

to be expressed The patient’s

of

and HBV of

in the

(3+) with cytoplasmic

in situ hybridization.

of

inflammatory

3+,

was also shown

of the hepatocytes

by nonisotopic

but

at 2.9

at that time of

showed

HBsAgpre-S,/pre-S,(3+,

antigen

HBeAg.

obtained

for cytomegalovirus

of the liver biopsy

in associa-

after discontinuation

specimen

flare showed

NO evidente

a mild hepatitic

occurring

leve1 that peaked

mg/dL)

A liver biopsy

the hepatitic sponse.

0.3-1.5

im-

for serum HBsAg,

He experienced

flare (AST, 102 IU/L;

HBIG

of HBIG pro-

HBcAg liver en-

zyme leve1 decreased within 1.5 times the upper limit of normal within 3 months of the initial hepatitic episode. He was in reasonably

good health

apart from occasional

for the following

mildly elevated

ase levels (al1 < 1.5 times the upper In April

15 months

serum aminotransferlimit of normal).

1992, 2 years after liver transplantation,

the pa-

tient presented again with bilateral ankle edema. The AST and ALT levels were mildly elevated at 98 and 93 IU/L, respectively, albumin low at 2.3 g/dL, bilirubin slightly elevated at 1.6 mg/dL,

and the prothrombin

time prolonged

to

13.2 seconds (control, 12 seconds). A serologie assessment documented that he was stil1 seropositive for HBsAg, anti-

Figure 1. Histological features of FCH in the patient. (A) Prominent hepatocyte ballooning (original magnification X250). (6) Extensive pericellular and bridging fibrosis (original magnification X90).

FCH IN HBV PRECORE MUTANT

September 1993

occasional

hepatocytes

in hepatocytes ing as shown staining

(
in serial biopsy

was found

The patient with

rapidly

bleeding

stain-

Strong

HBV DNA

of nearly

al1 hepato-

of his prothrombin

Immunohistochemical

few

(9 mg/dL)

and

time. He underwent

recipient

rizes the biochemical

hepatectomy.

and histological

Figure

features

a

a sec-

2 summa-

of the patient.

Laboratory Methods Serum

HBV

markers

and anti-HBe)

were

determined

immunoassays HBV

(Abbott

DNA

was achieved chain

by the respective

according

Sequencing

SSl85,

(anti-sense,

1 site 1) and SS186,

ECOR

codon

TGTAGGCATAAATTGGT-3’ with

(sense,

30 cycles of denaturation

nealing

at 50°C

minutes,

the

incubation

chain reaction

electrophoresis with

for 90 seconds,

and postcycle

The polymerase

at 94°C

followed

sequencing

by direct primer

TCAGAAGGCAAAAA-3’ cent-labeled

nucleotides.

ings were performed

an-

at 72’C

for 3

for 5 minutes.

was purified

by gel

sequencing

S-GGAAAGAAGcodon

1976-1954)

sequencer

Double-strand

to confirm

1778-1801)

bidirectional

(anti-sense,

and SS186 by the ABI 373 DNA

codon

at 72°C

JOL25,

num-

5’-GGAGGC-

for 90 seconds,

using

multiple

the mutation.

tection

of HBV DNA

hybridization

fluoressequenc-

It has been

HBsAg

1+

1+

---3+

Uvw HBcAg

2+

2+

-

Llvar HRaAg

-

-

- -

Uvw HBV DNA

t

+

---3t

HBsAg Antl-HBa HBV DNA

4t

-

-

t

-

+t

+

t

t

+

t

+t

+

+

+

+

-

+ +

t

t

Ckrh

kh.Klk changas Am+. “,.0tkm

and in situ hybridization

1%5%;

??

2 E 2 zi

z

. --s2

lOO--

5 ?? ’ 1 .

??

__ Y-4L

! 50.-

.$($_

:

.,I’----. _

L_/

.

z

i ??

ULN

.-*-.=.-.

-q--

.

OLT OL?

Dled

0, Apr 90

Oct 90

Apr 91

Oct 91

according

staining grading

3+, 30%60%;

to

of immunohiswas accomsystem:

0, nil;

and 4+ >60%).4

The patient in the present report illustrates two important mutant

points.

First,

recurrence

can be associated

of HBV

precore

with the development

FCH; second, hepatocytic

of

expression HBeAg is not an

essential element in the pathogenesis

of FCH.

Previous studies have shown that HBeAg is expressed in the hepatocytes

in patients

with FCH, suggesting

that the syndrome is a consequente

of infection

with

the wild-type HBV, although one cannot categorically rule out the presence of a “mixed” cases. Direct

sequencing

infection

in these

of the PCR product of this

patient’s HBV DNA allowed US to examine the possibility of a mixed infection. signal at position

The lack of the usual G

1896 characteristic

virus in the polymerase

chain reaction

gests that a virus having a mutation precore

mutant)

of the wild-type product

sug-

in this position

was the predominant

HBV

HBV form in the liver; however,

form of HBV in the liver was also

likely to be the precore mutant. The clinical course in the patient differed markedly

.

? 3

probe

Dein situ

Discussion

the predominant

FCH

*.

i

2+, 5%30%;

controls.

by nonisotopic

The grading

to the following

Al1

and nega-

the absente of both prominent nuclear HBcAg and hepatocytic HBeAg expression strongly suggests that

Mld bb”lW h.pa+ln.

positive

using digoxigenin-labeled

tochemical

per-

and in each instance

was performed method.’

antigens was

methods4s8

in the positive

described

according

HBV

HBcAg)

described

false negatives, positive

sent the predominant

f. 4t

N.D.

a

population in this patient. It may be argued that the HBV sequence obtained from serum may not repre-

4+

--st

and

a previously

(HBV Llwr

can detect

in 5% of the total

of the

HBeAg,

with appropriate

to avoid

was strongly

deregion

2484-2465,

extension

amplicon

tive controls

IL).

S-CCCACCTTA-

bering

is present

to previously

HBeAg

l+,

by polymerase

TGAGTCCAAGG-3’ system

was performed

enzyme

precore

the region

using the primers

staining

hybridization

HBV

that this method

detection

pre-S,,

according

plished

Chicago,

pre-S,,

formed

HBeAg,

to a previously

of the

by first amplifying

reaction

North

by nonisotopic

probe)

method.6

anti-HBs,

Laboratories,

was determined

(digoxigenin-labeled scribed

(HBsAg,

investigators of HBV which

HBV in the sample.’

10 weeks later and died of uncontrolled

during

by other

subpopulation

(HBsAg,

over the subsequent

hyperbilirubinemia

ond transplantation

HBcAg

in situ hybridization.

deteriorated

marked

prolongation

specimens.

shown

always occurred

for nuclear

in the cytoplasm

cytes (‘l+) by nonisotopic weeks

This finding

that were positive

903

Apr 92

Figure 2. Biochemical and histological profile of the patient. OLT, orthotopic liver transplantation; Cirrh, cirrhosis; ULN, upper limit of normal; ND, not done.

from previously reported patients with FCH. Specifically, he had a longer posttransplant period until the development

of FCIj

(23 months vs. 4-13

months in

the reported cases); also, a slower rate of clinical deterioration (10 weeks vs. 4-6 weeks in the reported cases) is notable. 2-1 There are a few possible reasons for these differences in the present cases compared with al1 previous reports. Firstly, the HBV precore mutant, although replication competent, may have a poorer Secondly, the immunosupreplication efficiency. pressive therapy (FK506 and prednisone) used in

904

FANG

GASTROENTEROLOGY

ET AL.

this patient

was different

in the previously affected

from the triple

reports.

the evolution

The use of FK506

of FCH. It should

in short-term primary hepatocyte corticoids, and not azathioprine have direct

enhancing

may have

be noted

that

5.

cultures, only glucoor cyclosporine A,

effect on HBV antigen exprespatient was on a similar

Because

regime

of glucocorticoids

ported

cases, the stimulatory

6.

as used in the previously

re-

effect of glucocorticoids

7.

was likely to be the same as in other cases, the possibility of a synergistic effect of the

various

agents

and

glucocorticoids

out. Finally,

the 6 months

have delayed

the clinical

the development Nonetheless, patient

used

the p resent

Sion.”

on HBV although

therapy

of HBIG

recurrence

with

the lack of HBeAg

expression

ballooning

of FCH.

This

alone

excessive

mouse

is sufficient

and cytopathic

changes

may

model

9.

element

conclusion

expression

8.

in this

is not an essential

the transgenic

sari et al. in which face antigens

prophylaxis of FCH.

for the development

be ruled

of HBV and hence

and the evolution

shows that HBeAg

accordance

cannot

is in

10.

of Chi-

of HBV sur-

to induce

extensive

in hepatocytes.”

References Iwatsuki S, Starzl TE, Todo S, Gordon RD, Esquivel CO, Tzakis AG, Makowka L, Marsh JW, Koneru B, Stieber A, Klintmalm G, Husberg B. Experience in 1,000 liver transplants under cyclosporine-steroid therapy: a survival report. Transplant Proc 1988;20:498-504. O’Grady JG, Smith HM, Davies SE, Daniels HM, Donaldson PT, Cohen AT, Portmann B, Alexander GJM, Williams R. Hepatitis B virus reinfection after orthotopic liver transplantation: serological and clinical implications. J Hepatol 1992; 14: 104- 1 1 1. Davies SE, Portmann B, O’Grady JG, Aldis PM, Chaggar K, Alexander GJM, Williams R. Hepatic histology following transplantation for chronic hepatitis B virus infection including a unique pattern of fibrosing cholestatic hepatitis. Hepatology 199 1; 13: 150-157. Lau JYN, Davies SE, Bain VG, O’Grady JG, Alberti A, Alexander

ll.

Vol. 105,

No. 3

GJM, Williams R. High leve1 expression of hepatitis 6 viral antigens in fibrosing cholestatic hepatitis. Gastroenterology 1992; 102:956-962. Benner KG, Lee RG, Keeffe EB, Lopez RR, Sasaki AW, Pinson CW. Fibrosing cytolytic liver failure secondary to recurrent hepatitis B after liver transplantation. Gastroenterology 1992; 103: 1307-1312. Naoumov NV, Lau JYN, Daniels HM, Alexander GJM, Williams R. Detection of HBV-DNA usinga digoxigenin probe: a quick method without loss of sensitivity. J Hepatol 199 1; 12:382-385. Raimondo G, Stemler M, Schneider R, Wildner G, Squadrito G, Will H. Latency and reactivation of a pre-core mutant hepatitis B virus in a chronically infected patient. J Hepatol 1990; 1 1:374380. Lau JYN, Bain VG, Davies SE, Alexander GJM. Williams R. Export of intracellular HBsAg in chronic hepatitis B virus infection IS related to viral replication. Hepatology 199 1; 14:4 16-42 1. Lau JYN, Naoumov NV, Alexander GJM, Williams R. Rapid detection of HBV DNA in liver tissue by in-situ hybridisation and its combination with immunohistochemistry for simultaneous detection of hepatitis B viral (HBV) antigens. J Clin Pathol 199 1;44:905-908. Lau JYN, Bain VG, Smith HM, Alexander GJM, Williams R. Modulation of hepatitis B viral antigen expression by immunosuppressive drugs in primary hepatocyte culture. Transplantation 1992;53:894-898. Chisari FV, Filippi P, Buras J, McLachalan A, Popper H, Pinkert CA, Palmiter RD, Brinster RL. Structural and pathological effects of synthesis of hepatitis 6 virus large envelope polypeptide in transgenie mice. Proc Natl Acad Sci USA 1987;84:6909-6913.

Received January 18, 1993. Accepted May 25, 1993. Address requests for reprints to: Johnson Y. N. Lau, M.D., Section of Hepatobiliary Diseases, University of Florida, P.O. Box 100214 JHMHC, Gainesville, Florida 32610. Supported in part by National Institutes of Health grant Al31918 (to F.Y.T.) and DSR-D-1591-92 grant (to J.Y.N.L.) from the Division of Sponsored Research of the University of Florida, Gainesville, Florida. The authors thank Drs. R. S. Tedder and B. Ferns, Middlesex Hospital, London, for their gifts of monoclonal antibodies to HBsAg and HBeAg; Prof. W. H. Gerlich and Dr. K. H. Heermann, Georg-August Universitat Göttingen, Göttingen, Germany, for their monoclonal antibody to pre-S, ; and Prof. A. Alberti, Clinica Medica II, Padova, Italy, for his monoclonal antibody to pre-S,.