Download Severe, Late-onset Graft-Versus-Host Disease in a Liver Transplant

Severe, Late-onset Graft-Versus-Host
Disease in a Liver Transplant Recipient
Documented by Chimerism Analysis
Marilyn S. Pollack, Kermit V. Speeg,
Natalie S. Callander, Cesar O. Freytes,
Alfredo A. Espinoza, Robert M. Esterl,
Gregory A. Abrahamian,
W. Kenneth Washburn, and Glenn A. Halff
ABSTRACT: A 52-year-old liver transplant recipient
presented 8 months after transplantation with oral thrush,
then 3 days later with oral ulcers and a diffuse rash, and
5 days later with an acutely reduced white blood cell
count, rash, fever, and diarrhea. Bone marrow biopsy
revealed severe aplasia. Although graft-versus-host disease
(GVHD) was considered, the late onset of these symptoms
was felt to render this etiology unlikely because GVHD
usually occurs 2 to 6 weeks after transplantation. All
potentially myelosuppressive medications were discontinued, and the patient was treated with high doses of
hematopoietic growth factors. Because his symptoms continued, chimerism analysis was performed, which indicated that 96% of the peripheral blood mononuclear cells
were of liver-donor origin. Ultimately, the patient underwent an allogeneic peripheral blood hematopoietic progenitor cell transplant from a human leukocyte antigen–
ABBREVIATIONS
BM
bone marrow
GVHD graft versus host disease
HLA
human leukocyte antigen
HPC
hematopoietic progenitor cells
INTRODUCTION
Graft-versus-host disease (GVHD) occurs when a transplant donor’s T lymphocytes differentiate into effector
From the Departments of Pathology (M.S.P), Medicine (K.V.S., N.S.C.,
C.O.F., A.A.S), and Surgery (R.M.E., G.A.A., W.K.W., G.A.H.), University of Texas Health Science Center, San Antonio, TX, USA.
Address reprint requests to: Dr. Marilyn S. Pollack, Department of
Pathology, University of Texas Health Science Center, 7703 Floyd Curl
Drive, San Antonio, TX 78229; Tel: (210) 567-5698; Fax: (210)
358-0777; E-mail: [email protected].
Received June 29, 2004; revised September 24, 2004; accepted September
24, 2004.
Human Immunology 66, 28 –31 (2005)
© American Society for Histocompatibility and Immunogenetics, 2005
Published by Elsevier Inc.
identical brother, but he died 5 days after transplantation
of overwhelming Candida kruseii infection. To our knowledge, this is the first chimerism-analysis– documented
case of severe acute GVHD presenting so late after liver
transplantation. It is of note that the patient had no
known risks for GVHD in that he was relatively young
and shared only one major human leukocyte antigen with
his donor. Consideration should be given to GVHD as a
cause of bone marrow aplasia at any time after organ
transplantation. Storage of cell pellets from all transplant
recipients and donors is highly recommended to facilitate
the diagnostic evaluation.
Human Immunology 66,
28 –31 (2005). © American Society for Histocompatibility and Immunogenetics, 2005. Published by Elsevier Inc.
KEYWORDS: liver transplantation; graft versus host
disease; chimerism analysis; HLA
MC
PB
WBC
mononuclear cell
peripheral blood
white blood cells
cells that mount an immune response against recipient
tissues. This is a frequent complication of allogeneic
hematopoietic progenitor cell (HPC) transplantation because of the planned infusion of large numbers of immunocompetent cells into an immunocompromised host. In
solid organ transplantation, GVHD is an infrequent but
serious complication and generally only occurs for transplants involving organs with relatively large numbers of
passenger lymphocytes, notably for liver and intestine
transplantation. Skin and gut manifestations of GVHD
in liver transplant recipients resemble those observed in
0198-8859/05/$–see front matter
doi:10.1016/j.humimm.2004.09.014
GVHD Documented by Chimerism Analysis
HPC transplant recipients, including skin rash and diarrhea. However, liver transplant recipients with GVHD
typically lack evidence of liver dysfunction and instead
have severe pancytopenia and bone marrow (BM) aplasia
not usually seen in HPC transplant recipients. The diagnosis of GVHD in organ transplant recipients is
largely a clinical one, but recently, more sophisticated
testing that allows the detection of donor organ– derived
cells in affected tissue sites can confirm the clinical
suspicion and prevent institution of inappropriate therapy. For example, a skin rash with infiltrating donor
lymphocytes should probably not be treated as a druginduced toxicity with concomitant reduction of a particular drug.
The primary risk factor for the development of
GVHD in organ transplant recipients is considered to be
a relatively low degree of human leukocyte antigen
(HLA) mismatching between the donor and recipient,
such as in cases of related-donor (usually parent to child)
liver transplants or donors homozygous for antigens at
most or all HLA loci. A recent meta-analysis [1] of
factors increasing the risk for GVHD concluded that in
addition to low HLA mismatching, a relatively large age
discrepancy (⬎40 years) between donor (younger) and
recipient (older) and recipient age over 65 years were
contributing risk factors, presumably as a result of loss of
immune responsiveness (against donor lymphocytes) as a
function of age. Organ transplant–related GVHD has
only been documented to occur for the first time within
the first few months after organ transplantation [1],
when immunosuppression of the recipient is at its highest level and presumably because donor lymphocytes
surviving beyond that time have become tolerant to
recipient mismatches. We describe here a liver transplant
patient presenting for the first time 8 months after
transplantation with clinical and subsequent confirmatory chimerism evidence of GVHD.
CASE REPORT
This case involves a 52-year-old Hispanic man, blood
type O positive, who received an orthotopic liver transplant for end-stage liver disease from alcoholic cirrhosis.
The donor was a 53-year-old woman, blood type O
positive, who died from a subarachnoid hemorrhage. She
had normal liver enzymes. All pretransplant crossmatch
tests were negative. The patient underwent an uneventful liver transplantation with typical preparation of the
organ, including perfusion with recipient blood, and was
discharged 7 days after surgery. According to the routine
protocols at our institution, he did not receive any
antilymphocyte induction therapy, and his immunosuppression over the next 8 months consisted of low-dose
tacrolimus, mycophenolate mofetil (tapered off after 14
29
weeks), and prednisone (5 mg daily). He had no episodes
of rejection and had normal liver enzymes, with gradual
resolution of the renal insufficiency that had predated his
transplant.
At 8 months after transplant, the patient presented
with oral thrush, which was treated with fluconazole.
Three days later, he presented with a sore throat, oral
ulcers, and a diffuse rash; fluconazole therapy was
stopped. His white blood cell (WBC) count, which had
been normal, was 2500/␮l and then acutely dropped over
the next 5 days to 0.1 ␮l/ml, accompanied by severe
anemia and thrombocytopenia. The patient was admitted
with a 1-day history of temperature to 40°C, diffuse
maculopapular rash, diarrhea, and abdominal pain. Blood
cultures were negative, as were tests for cytomegalovirus.
A BM biopsy on the second hospital day revealed no
aspirable elements and an overall cellularity of 10%.
Special stains for mycobacteria and fungi were negative.
Although GVHD was considered, the majority of the
staff believed that the long interval between transplantation and the patient’s presentation made GVHD unlikely. Instead, it was believed that the rash and marrow
aplasia were a result of drug therapy including valganciclovir and Septra. Both of those drugs were discontinued, and he was treated with broad-spectrum antibiotics
and large doses of erythropoietin and Filgrastim. Because
he clinically stabilized, he was discharged to home with
oral antibiotics and growth factors.
The patient was subsequently readmitted 5 days later
with recrudescence of the rash and fever. Meanwhile, a
skin biopsy that had been performed during his first
admission was read by a hematopathologist as consistent
with grade II GVHD. Chimerism analysis of separated
peripheral blood (PB) mononuclear cells (MNCs) and
granulocytes and total BM nucleated cells (the sample
was too acellular to analyze separated BM MCs) was then
performed. Stored, pretransplant WBC pellets from both
the recipient and donor were available for comparison.
DNA from all samples was extracted by means of a
standard Qiagen protocol. The chimerism analysis was
done with an ABI sequencer with a Promega PowerPlex
System in which ten polymorphic, single tandem repeat
system alleles and the X versus Y chromosome marker
Amelogenin were simultaneously amplified and labeled
with different fluorescent dyes. Polymerase chain reaction products with different size (repeat number) polymorphisms were then separated from each other by their
rate of migration in a capillary gel. The results indicated
that all systems tested were informative in one or both
directions (Table 1) and that virtually all (96%) of the
PB MCs (largely lymphocytes) were of liver donor origin
(Figure 1, third panel). The PB granulocyte fraction was
75% donor, 30% recipient (data not shown). The BM
total nucleated cells appeared to be only 30% of donor
30
M.S. Pollack et al.
TABLE 1 Recipient pretransplant and donor STR
system alleles
STR system
Recipient pretransplant alleles
Donor alleles
D3S1358
VWA
D16S539
D2S1338
D8S1170
D21S11
D18S51
D19S433
THO1
FGA
Amelogenin
15, 18
17, 19
11, 12
16, 23
13, 14
31.2
12, 17
13, 14
7, 9.3
21, 25
X, Y
16, 18
14, 16
10, 11
17, 24
13, 15
30, 34.2
17, 20
14
6, 7
22, 24
X, X
origin (Figure 1, last panel), but because the BM was so
aplastic, it was presumed that the sample contained more
recipient-derived stromal cells than cells of hematopoietic lineage. Graft-versus-host disease was then diagnosed as the certain cause of the BM aplasia. Surprisingly, an analysis of the patient and donor HLA
phenotypes (Table 2) indicated only a single major antigen match (HLA-B35), with two mismatches for
HLA-A, one for HLA-B, two for HLA-C, two for HLADR, and two for HLA-DQ.
A review of the literature indicated that both increasing
and decreasing immunosuppression [1–3] have been used
successfully in treating organ-transplant-related acute
GVHD, although outcomes have generally been poor,
regardless of treatment, with a mortality that exceeded
75% [1]. In this case, a course of OKT3 was provided as
therapy for the GVHD. Although OKT3 reduced the total
lymphocyte count even further, there was no recovery of
the total WBC count, and repeat chimerism analysis indicated that the proportion of donor MCs remained the
same. Because the therapy with OKT3 failed to control the
GVHD, the patient underwent an allogeneic PB HPC
transplant from an HLA-identical sibling after conditioning with cyclophosphamide and antithymocyte globulin, a
combination routinely used for patients undergoing HPC
transplantation for aplastic anemia. However, despite aggressive blood product support, total parenteral nutrition,
and broad-spectrum antibiotic and antifungal therapy, the
patient by this time was severely debilitated and had been
pancytopenic for almost 2 months. He developed a disseminated Candida kruseii infection and died on the fifth
day after stem cell transplantation of multiorgan failure
with no evidence of marrow recovery.
DISCUSSION
Liver transplant recipients who develop acute, rapidly progressing GVHD with large numbers of circulating donor
lymphocytes generally do so 2 to 6 weeks after transplan-
tation [1]. This case report illustrates that acute GVHD
can manifest for the first time many months after liver
transplantation. No precipitating cause for its occurrence
could be clearly identified. There was no change in the
patient’s immunosuppressive regimen, he did not receive
any blood transfusions, and he had no evidence for any
systemic inflammatory response. The lack of any rejection
episodes was not indicative of unusual hypoimmunity because approximately 90% of the more than 120 liver
transplant recipients we treat per year at our institution
also do not experience any rejection episodes, but this is
our first case of documented GVHD. There is, however, a
theoretic possibility that, as occurs in many autoimmune
diseases, the immune response to his local fungal infection
activated donor lymphocytes, which then became sensitized to shared epitopes present in noninfected recipient
FIGURE 1 Chimerism analysis 8.5 months after transplant
(during severe bone marrow (BM) aplasia). Results for four of
the 11 systems tested are shown in the first panel using the
patient’s pretransplant peripheral blood (PB) DNA; second
panel, donor PB DNA; third panel, the patient’s 8.5-month
posttransplant PB mononuclear cell DNA (96% donor); and
last panel, the patient’s 8.5-month posttransplant BM aspirate
total nucleated cell DNA (30% donor). D ⫽ donor-specific
alleles; R ⫽ recipient-specific alleles; D, R ⫽ shared alleles.
For allele numbers for each system, see Table 1. Results for the
other seven systems were similar.
GVHD Documented by Chimerism Analysis
TABLE 2 Recipient and donor HLA types
Subject demographics
HLA phenotype
Recipient (52-year-old man)
A2, 29; B13 (Bw4), 35(Bw6); Cw4,
w6; DR4, 7; DR53; DQ2, 8
A1, 3; B8 (Bw6), 35 (Bw6); Cw4, w7;
DR1, 17 (3); DR52; DQ2, 5
Donor (53-year-old woman)
Abbreviation: HLA ⫽ human leukocyte antigen.
tissues. Whatever the precipitating cause, the resulting
marrow aplasia evidently stimulated new blood cell formation from donor hematopoietic stem cells that had been
transplanted along with the graft, because in addition to
the donor lymphocytes, 75% of the circulating granulocytes at the time of GVHD diagnosis were of donor origin.
Other reports of GVHD occurring late after transplantation appear to involve more easily treatable chronic
GVHD rather than acute GVHD. One previous report [2]
described recurrent GVHD in two pediatric liver transplant recipients at approximately 11 and 7 months after
transplantation that had first manifested at approximately
3 and 4 months after transplantation, respectively. For
these patients, only about 3% donor cells were detected in
the PB, and both patients responded to temporary discontinuation of immunosuppressive therapy. Dunn et al. [3]
described another child who presented 5 years after liver
transplantation with intestinal and skin symptoms consistent with GVHD. Her PB revealed (only) 1% male cells,
but because she had received six of 16 units of unirradiated
blood from male donors, the diagnosis of GVHD could not
be attributed to liver donor lymphocytes; the patient
quickly recovered after treatment with a small pulse of
steroids. It should also be noted that a small degree of
chimerism is often found after organ transplant for recipients who do not experience any symptoms of GVHD [4].
The patient described in our case report had none of the
risk factors previously associated with GVHD presenting
in the first few weeks after liver transplant in that there
was a high degree of HLA mismatching and the recipient
was relatively young. However, two other risk factors
might be implicated in the development of his GVHD.
First, the donor was found to have an HLA phenotype
(Table 2) consistent, because of significant linkage disequilibrium, with having the HLA haplotype that is most
frequently documented to confer a higher than normal risk
for hyperimmune responses that result in autoimmune
diseases: HLA-A1, B8, DR17 (DR3), DQ2 [5, 6]. In this
regard, it is intriguing that one of the two patients described by Pinna et al. as having recurrent GVHD many
months after transplant also had a donor with this haplotype ([2] (Table 1)) (population frequency less than 9%).
Perhaps the persistence after transplantation of immunoresponsive donor lymphocytes is associated with their his-
31
tory of hyperimmune responses. In addition, as a woman,
the liver donor for our patient would also be predicted to
have relatively high immune reactivity well known, for
example, to cause a greater risk of GVHD for recipients of
female HPC transplants [7], possibly derived from exposure to alloantigens during pregnancy. Studies of other
liver transplant patients with GVHD that look for multiparous female versus male donor status should be considered to see whether that may be generally considered an
additional risk factor for the development of GVHD after
liver transplantation. In any case, histocompatibility laboratories are strongly encouraged to save test material from
all organ transplant recipients and donors, even if their
transplant programs do not routinely request HLA typing
for liver transplant recipients. The cost for long-term storage of cell pellets is relatively low, and a reference laboratory can be used if chimerism testing is not available on
site. A rapid diagnosis of GVHD will allow early therapy,
giving patients a better chance for recovery.
ACKNOWLEDGMENTS
We thank Julie B. Forman and Laura M. McNeish for expert
technical assistance.
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