Pediatr Blood Cancer 2007;49:93–98

BRIEF REPORTImmunotherapy for Severe Aplastic Anemia Following Orthotopic

Liver Transplantation in Children

Rosa Sanchez, MD,1,3* Philip Rosenthal, MD,1,2,4,5 and Robert Goldsby, MD1,3

INTRODUCTION

Severe aplastic anemia is a devastating, although well recog-

nized, complication after orthotopic liver transplantation for

fulminant hepatitis [1–3]. The incidence of bone marrow failure

following fulminant hepatitis ranges from 10.7% to 33% in various

case reports [1,2,4]. Severe aplastic anemia was observed in 28% of

patients who underwent liver transplantation for fulminant non-A,

non-B, and non-C hepatitis in one case series [2]. Bone marrow

failure can present concurrently with liver failure or within weeks

following orthotopic liver transplantation [2]. If a matched-related

donor is available, a bone marrow or peripheral blood stem cell

transplant is the primary treatment choice for severe aplastic

anemia. Immunosuppressive therapy is effective in treating severe

aplastic anemia in patients who lack a histocompatible donor or

are poor candidates for stem cell transplant. Patients with severe

aplastic anemia following orthotopic liver transplantation have

been successfully treated with bone marrow transplantation, if a

histocompatible donor is available, or with immunotherapy alone

[2,5,6]. We describe two children who developed severe aplastic

anemia following orthotopic liver transplantation for fulminant non-

A, non-B, and non-C hepatitis. Both patients were switched from

cyclosporine to tacrolimus due to cyclosporine toxicity, and both

maintained adequate hematopoeisis on tacrolimus.

CASE ONE

A previously healthy 6-year-old Caucasian girl presented with

jaundice and acute hepatitis. The patient’s laboratory findings on

admission are summarized in Table I. Serology testing for hepatitis

(A, B, and C) and other infectious causes, a metabolic and

autoimmune work-up did not reveal an inciting cause for acute

hepatitis. Her liver function steadily deteriorated in spite of

aggressive supportive care. Approximately 1 week after admission,

she developed grade 4 encephalopathy requiring mechanical

ventilation and underwent a cadaveric orthotopic liver transplant.

She did well post-operatively on an immunosuppression regimen

of mycophenolate mofetil, cyclosporine, and methylprednisolone.

She also received anti-viral (ganciclovir), Pneumocystis carinii

pneumonia (trimethoprim-sulfamethoxazole), and anti-fungal (nys-

tatin) prophylaxis. One week following transplant, the patient

developed anemia (hemoglobin 8.8 g/dl) and thrombocytopenia

(platelet count 28� 109/L). A bone marrow examination revealed a

hypocellular marrow with 20% cellularity with mixed hematopoi-

esis. She continued with persistent myelosuppression despite

discontinuation of mycophenolate mofetil, ganciclovir, and tri-

methoprim-sulfamethoxazole (Fig. 1). Two weeks later, a second

bone marrow examination revealed a markedly hypocellular

marrow (5% cellularity) consistent with evolving aplastic anemia.

It was decided to treat her severe aplastic anemia with immuno-

therapy consisting of anti-thymocyte globulin (ATG) (15 mg/kg/

day) for 10 days, methylprednisolone (2 mg/kg/day) during the anti-

thymocyte globulin treatment, and ongoing cyclosporine (9–12 mg/

kg/day) twice daily. The corticosteroids were then tapered off over

8 months per the liver transplant protocol.

One month after starting cyclosporine, she experienced undesir-

able side effects of hypertrichosis, hypertension, and difficulty

maintaining a therapeutic drug level between 150–200 mg/L. Her

calcineurin inhibitor therapy was switched from cyclosporine to

tacrolimus, which she tolerated well. While she did experience a

second period of myelosuppression, her bone marrow recovered

after decreasing the dose of mycophenolate mofetil and changing

trimethoprim-sulfamethoxazole to dapsone (Figs. 1, 3, 4). The

patient continues with full bone marrow recovery and normal liver

function approximately 2½ years following cadaveric orthotopic

liver transplant for idiopathic fulminant hepatitis. She continues to

receive tacrolimus daily to prevent liver graft rejection.

Severe aplastic anemia is a well-recognized complication offulminant non-A, non-B, and non-C hepatitis requiring orthotopicliver transplantation. The first line of therapy for cure in the treatmentof aplastic anemia is a histocompatible bone marrow transplant.Immunosuppressive therapy is also effective if a histocompatibledonor is not available. We describe two children who developed

severe aplastic anemia following orthotopic liver transplant whoachieved bone marrow recovery with a single course of anti-thymocyte globulin, solumedrol, and adjustments to their immuno-suppressive therapy for prevention of liver allograft rejection. PediatrBlood Cancer 2007;49:93–98. � 2006 Wiley-Liss, Inc.

Key words: aplastic anemia; children; cyclosporine; hepatitis; liver transplantation; tacrolimus

� 2006 Wiley-Liss, Inc.DOI 10.1002/pbc.20718

——————1Department of Pediatrics, University of California, San Francisco,

California; 2Department of Surgery, University of California, San

Francisco, California; 3Division of Hematology-Oncology, University

of California, San Francisco, California; 4Divisions of Gastro-

enterology, Hepatology, and Nutrition, University of California, San

Francisco, California; 5Division of Liver Transplantation, University

of California, San Francisco, California

*Correspondence to: Rosa Sanchez, UCSF, Department of Pediatrics,

505 Parnassus Ave. M649, Pediatric Hematology/Oncology, San

Francisco, CA 94143-0106. E-mail: rsanchez@itsa.ucsf.edu

Received 25 May 2005; Accepted 31 October 2005

CASE TWO

A 5½-year-old caucasian boy presented with acute liver

dysfunction and coagulopathy. Laboratory findings on admission

are summarized in Table I. The hepatitis serologies (hepatitis A, B,

and C), autoimmune, infectious, and metabolic work-up was

negative. A bone marrow examination performed 2 days after

admission to evaluate for evolving aplastic anemia revealed a 45%

cellularity with mixed hematopoiesis. In spite of supportive

measures, he developed altered mental status and deteriorating

liver function, and 6 days after admission underwent a living-related

liver transplant on an urgent basis. His post-operative course was

complicated by graft failure secondary to hepatic artery thrombosis

and severe pancytopenia. Post-operatively, his immunosuppressive

regimen consisted of mycophenolate mofetil, cyclosporine, and

methylprednisolone. One week after transplant, mycophenolate

mofetil, trimethoprim-sulfamethoxazole, and ganciclovir were

discontinued secondary to persistent myelosuppression (Fig. 2).

Approximately 2 weeks after transplant, a second bone marrow

examination demonstrated a hypoplastic bone marrow with 10%

cellularity, mostly lymphocytes and plasma cells. Due to graft

failure, he was re-listed for a liver transplant on an emergency basis,

and 18 days after the first transplant, he received a cadaveric

orthotopic liver without any complications. Post-operatively, he

received anti-viral, anti-fungal, and Pneumocystis carinii pneumo-

nia prophylaxis. Immunotherapy for severe aplastic anemia was

started 2 days after the second liver transplant and included anti-

thymocyte globulin (15 mg/kg/day) and methylprednisolone (2 mg/

kg/day) for 10 days, and cyclosporine (9–12 mg/kg/day).

Approximately 3 weeks after the second liver transplant, he

developed altered mental status. Radiologic evaluation of the brain

with CT and MRI did not reveal any intracranial ischemia or bleed.

His altered mental status was attributed to cyclosporine toxicity, and

it was held temporarily until his neurologic symptoms improved. A

week later, cyclosporine was restarted, initially at a small dose that

was then increased to full dose. One week after restarting the

cyclosporine, he underwent a liver biopsy to evaluate rising serum

transaminase levels that were consistent with moderate acute

cellular rejection of the graft. He was switched from cyclosporine to

tacrolimus. Despite changing to tacrolimus, his bone marrow recovery

was sustained (Figs. 2–4). Two years after orthotopic liver transplant

for idiopathic fulminant hepatitis and treatment with immunotherapy

for severe aplastic anemia, the patient’s liver function and bone

marrow are normal.

DISCUSSION

Severe aplastic anemia after liver transplantion for fulminant

non-A, non-B, and non-C hepatic failure is a serious and relatively

common complication. Successful treatment of severe aplastic

anemia following orthotopic liver transplantion with immunother-

apy has been reported [6]. We describe two children with severe

aplastic anemia following orthotopic liver transplant successfully

treated with combination therapy of anti-thymocyte globulin,

methylprednisolone, and adjustments to routine calcineurin inhibi-

tors. The effectiveness of treating severe aplastic anemia with

immunotherapy alone in patients already on immunosuppressive

therapy for prevention of rejection is concerning.

The use of immunotherapy as a treatment modality for severe

aplastic anemia was discovered when bone marrow recovery was

Pediatr Blood Cancer DOI 10.1002/pbc

TABLEI.

Characteristics

ofPatients

PresentingWithAcute

Hepatitis

Cas

eA

ge/

sex

TB

ili

(mg

/dl)

AS

T(U

/L)

AL

T(U

/L)

PT

(sec

)aP

TT

(sec

)

Fib

rin

og

en

(mg

/dl)

Hg

b(g

/dl)

Plt

(�1

09/L

)W

BC

(�1

09/L

)A

NC

(�1

09/L

)

n¼

0.3

–1

.3n¼

16

–4

1n¼

12

–5

9n¼

11

.1–

14

.5n¼

24

.5–

35

.2n¼

17

5–

43

3n¼

11

.4–

15

.5n¼

14

0–

45

0n¼

4.5

–1

5.5

n¼

1.5

–8

.5

16

-yea

r-o

ld/F

21

.41

73

21

86

43

0.3

47

.67

81

3.4

21

35

.43

.68

25

-yea

r-o

ld/M

15

.41

58

31

34

84

6.4

50

.11

16

9.6

28

6.7

5.0

2

n,n

orm

alval

ues

for

age.

94 Sanchez et al.

noted during the conditioning regimen for bone marrow transplan-

tation consisting of anti-lymphocyte globulin (ALG) [7]. The

addition of cyclosporine achieved better response rates of bone

marrow recovery than with anti-lymphocyte globulin therapy alone.

A multi-center randomized clinical trial comparing treatment with

ALG and methylprednisolone (control group) with ALG, methyl-

prednisolone, and cyclosporine, revealed a 31% response rate in the

control group at 6 months, as compared to a 65% response in the

treatment group receiving cyclosporine [8]. A single course

(10 days) of anti-thymocyte globulin (ATG) is as effective for

severe aplastic anemia as two courses [9]. One course of ATG,

combined with cyclosporine and methylprednisolone, resulted in a

response rate of 67% [9]. A 70% response rate was observed with

two sequential courses of ATG therapy combined with cyclosporine

and methylprednisolone [10].

Itterbeek et al. described a case report and review of the literature

on aplastic anemia after transplantation for non-A, non-B, non-C

fulminant hepatic failure [11]. A 20-year-old female who developed

aplastic anemia 14 weeks after liver transplantation achieved bone

marrow recovery after ATG, tacrolimus, and high-dose steroids

[11]. Spontaneous recovery of aplastic anemia under maintenance

immunosuppression for liver transplantation has been described, but

increased immunosuppression with ATG may reverse the aplastic

anemia [3,11,12]. The two cases presented in this report were treated

with one course of ATG and high-dose steroids in addition to their

maintenance immunosuppression.

According to the response criteria for treatment of aplastic

anemia proposed by Camitta [13], Case 1 had a complete response

after ATG and high-dose steroids. The response criteria are based on

two blood count results drawn 4 weeks apart while not receiving

colony-stimulating factors. The hemoglobin should be normal for

age, the absolute neutrophil count greater than 1.5� 109/L, and the

platelet count greater than 150� 109/L [13] (Figs. 1, 3, 4). Case 2

had a complete response after one course of ATG and high-dose

steroids. The absolute neutrophil count was greater than 1.5� 109/L

on blood count results drawn 4 weeks apart. His platelet count

took longer to recover (Figs. 2–4). The most recent count was

200� 109/L.

In patients with cyclosporine toxicity or other reasons to avoid

cyclosporine, tacrolimus may be considered as an alternative choice

of immunotherapy for the treatment of aplastic anemia. Tacrolimus

and cyclosporine are both calcineurin inhibitors. Both drugs are

known to inhibit interleukin-2 (IL-2) production and block the

proliferation of T-cells. They may treat severe aplastic anemia by

the same mechanism [14]. An expanded population of cytotoxic

T-lymphocytes likely mediate myelosuppression. These cells

produce inhibitory cytokines, interferon-gamma, and tumor necro-

sis factor-alpha, which are capable of suppressing both progenitor

cell growth and induce programmed cell death to CD34þ cells [14].

Cyclophosphamide, a potent immunosuppressive agent, has

been successful in restoring hematopoiesis in patients with severe

aplastic anemia [14,15]. It is used as a conditioning agent in patients

Pediatr Blood Cancer DOI 10.1002/pbc

Fig. 1. Case 1 received an orthotopic liver transplant on day 9. TMP-SMX*, ganciclovir, and MMF{were given on days 9–22. ATG{ and high dose

steroids were given on days 38–47. On day 67, cyclosporine was switched to tacrolimus. On days 108–126, TMP-SMX* was switched to dapsone,

and MMF{ was decreased. {TMP-SMX (trimethoprim-sulfa-methoxazole) {MMF (mycophenolate mofetil) {ATG (anti-thymocyte globulin).

Immunotherapy Following Liver Transplantation 95

Pediatr Blood Cancer DOI 10.1002/pbc

Fig. 3. The platelet counts for Case 1 recovered after ATG{ and high-dose steroids given on days 38–47. A rise in the platelet count was noted

after decreasing MMF{ and switching TMP-SMX* to dapsone on days 108–126. Case 2 received ATG{ and high-dose steroids on days 25–35.

The platelet counts for Case 2 took longer to recover. {ATG (antithymocyte-globulin) {MMF (mycophenolate mofetil). *Trimethoprim-

sulfamethoxazole.

Fig. 2. Case 2 received liver transplant #1 on day 6 and liver transplant #2 on day 24. TMP-SMX*, ganciclovir and MMF{ were given on days 6–

12. On days 25–35, ATG{ and high-dose steroids were given. On day 28, TMP-SMX* and ganciclovir were restarted. On day 36, TMP-SMX* was

switched to dapsone, and ganciclovir was decreased. On day 41, cyclosporine was discontinued, and MMF{was restarted. On day 50, tacrolimus was

started. *TMP-SMX (trimethoprim-sulfa-methoxazole) {MMF (mycophenolate mofetil) {ATG (anti-thymocyte globulin).

96 Sanchez et al.

undergoing bone marrow transplantation. There are case reports of

complete restoration of bone marrow function with cyclopho-

sphamide despite failure of allogenic bone marrow engraftment

[16–18]. A randomized clinical trial conducted at the National

Institutes of Health compared ATG/cyclosporine to high-dose

cyclophosphamide and cyclosporine as initial treatment in patients

with severe aplastic anemia [19]. The trial was terminated early due

to excessive toxicities in the group of patients that received

cyclophosphamide [20].

Either a matched-related bone marrow transplant, or ATG/

cyclosporine with high-dose steriods have been reported as

treatment options for acquired aplastic anemia after orthotopic

liver transplantation for fulminant non-A, non-B, and non-C

hepatitis [3,5,6]. Tacrolimus may be substituted for cyclosporine

for the treatment of severe aplastic anemia in children who

experience cyclosporine related undesirable side effects.

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and septra was switched to dapsone. The reticulocyte count (retic*) on days 15, 30, and 126 were 80, 4, and 198, respectively. Case 2 received ATG{

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globulin) {MMF (mycophenolate mofetil) *retic (reticulocyte count) normal value¼ (26–110� 109/L).

Immunotherapy Following Liver Transplantation 97

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