Spontaneous Autologous Graft-versus-Host Disease in Plasma Cell Myeloma Autograft Recipients: Flow Cytometric Analysis of Hematopoietic Progenitor Cell Grafts

ArticleinBiology of blood and marrow transplantation: journal of the American Society for Blood and Marrow Transplantation 17(7):970-8 · March 2011with47 Reads
Impact Factor: 3.40 · DOI: 10.1016/j.bbmt.2011.03.005 · Source: PubMed
Abstract

Nine plasma cell myeloma patients spontaneously developed histologically proven autologous graft-versus-host disease (GVHD) limited predominantly to the gastrointestinal tract within 1 month of initial autologous hematopoietic cell transplantation (AHCT) using high-dose melphalan conditioning. All recipients responded promptly to systemic and nonabsorbable oral corticosteroid therapy. All patients previously received systemic therapy with thalidomide, lenalidomide, or bortezomib before AHCT. Using enzymatic amplification staining-enhanced flow cytometry, we evaluated expression of selected transcription regulators, pathway molecules, and surface receptors on samples of the infused hematopoietic cell grafts. We demonstrated significantly enhanced expression of GATA-2, CD130, and CXCR4 on CD34(+) hematopoietic progenitor cells of affected patients compared with 42 unaffected AHCT controls. These 3 overexpressed markers have not been previously implicated in autologous GVHD. Although we did not specifically evaluate T cells, we postulate that exposure over time to the various immunomodulating therapies used for induction treatment affected not only the CD34(+) cells but also T cells or relevant T cell subpopulations capable of mediating GVHD. After infusion, the affected hematopoietic progenitor cells then encounter a host that has been further altered by the high-dose melphalan preparative regimen; such a situation leads to the syndrome. These surface markers could be used to develop a model to predict development of this syndrome. Autologous GVHD potentially is a serious complication of AHCT and should be considered in plasma cell myeloma patients with otherwise unexplained gastrointestinal symptoms in the immediate post-AHCT period. Prompt recognition of this condition and protracted treatment with nonabsorbable or systemic corticosteroids or the combination may lead to resolution.

Full-text

Available from: David Kaplan, Mar 18, 2014
Spontaneous Autologous Graft-versus-Host Disease
in Plasma Cell Myeloma Autograft Recipients: Flow
Cytometric Analysis of Hematopoietic Progenitor
Cell Grafts
Hillard M. Lazarus,
1
Scott R. Sommers,
1
Lisa M. Arfons,
2
Pingfu Fu,
3
S . A. Ataergin,
4
N. M. Kaye,
5
F. Liu,
5
Tamila L. Kindwall-Keller,
1
Brenda W. Cooper,
1
Mary J. Laughlin,
1,
*
Richard J. Creger,
1
Paul M. Barr,
1,
Stanton L. Gerson,
1
David Kaplan
5,6
Nine plasma cell myeloma patients spontaneously developed histologically proven autologous graft-versus-
host disease (GVHD) limited predominantly to the gastrointestinal tract within 1 month of initial autologous
hematopoietic cell transplantation (AHCT) using high-dose melphalan conditioning. All recipients responded
promptly to systemic and nonabsorbable oral corticosteroid therapy. All patients previously received sys-
temic therapy with thalidomide, lenalidomide, or bortezomib before AHCT. Using enzymatic amplification
staining-enhanced flow cytometry, we evaluated expression of selected transcription regulators, pathway
molecules, and surface receptors on samples of the infused hematopoietic cell grafts. We demonstrated sig-
nificantly enhanced expression of GATA-2, CD130, and CXCR4 on CD34
1
hematopoietic progenitor cells
of affected patients compared with 42 unaffected AHCT controls. These 3 overexpressed markers have not
been previously implicated in autologous GVHD. Although we did not specifically evaluate T cells, we pos-
tulate that exposure over time to the various immunomodulating therapies used for induction treatment af-
fected not only the CD34
1
cells but also T cells or relevant T cell subpopulations capable of mediating
GVHD. After infusion, the affected hematopoietic progenitor cells then encounter a host that has been fur-
ther altered by the high-dose melphalan preparative regimen; such a situation leads to the syndrome. These
surface markers could be used to develop a model to predict development of this syndrome. Autologous
GVHD potentially is a serious complication of AHCT and should be considered in plasma cell myeloma pa-
tients with otherwise unexplained gastrointestinal symptoms in the immediate post-AHCT period. Prompt
recognition of this condition and protracted treatment with nonabsorbable or systemic corticosteroids or
the combination may lead to resolution.
Biol Blood Marrow Transplant 17: 970-978 (2011) Ó 2011 American Society for Blood and Marrow Transplantation
KEY WORDS: Autologous hematopoietic progenitor cell transplant, Autologous graft-versus-host disease,
Plasma cell myeloma
INTRODUCTION
Autologous hematopoietic cell transplantation
(AHCT) is an integral part of standard therapy
for plasma cell myelo ma patients, and this disorder is
the most common indication for AHCT in North
America [1-3]. Although allogeneic hematopoietic
From the
1
Department of Medicine, Division of Hematology/
Oncology, Case Comprehensive Cancer Center, University
Hospitals Case Medical Center, Cleveland, Ohio;
2
Depart-
ment of Medicine, Division of Hematology/Oncology, Louis
Stokes Cleveland VAMC, Cleveland, Ohio;
3
Department of
Epidemiology and Biostatistics, Case Western Reserve Uni-
versity, Cleveland, Ohio;
4
Department of Medical Oncology
and Bone Marrow Transplantation Unit, Gulhane (GATA)
Facult y of Medicine, Ankara, Turkey;
5
Pathfinder Biotech,
Cleveland, Ohio; and
6
Department of Pathology, University
Hospitals Case Medical Center, Cleveland, Ohio.
Financial disclosure: See Acknowledgments on page 976.
* Current address: Department of Medicine, Division of Hematology/
Oncology, University of Virginia, Charlottesville, VA 29908.
Current address: Department of Medicine, Division of Hematology/
Oncology, University of Rochester, Rochester, NY 14642.
Correspondence and reprint requests: Hillard M. Lazarus, MD,
FACP, Department of Medicine, University Hospitals Case
Medical Center, 11100 Euclid Avenue, Cleveland, OH 44106
(e-mail: hillard.lazarus@case.edu).
Received January 17, 2011; accepted March 10, 2011
Ó 2011 American Society for Blood and Marrow Transplantation
1083-8791/$36.00
doi:10.1016/j.bbmt.2011.03.005
970
Page 1
cell transplantation may result in a higher rate of
molecular remission via an allogeneic effect, that is,
graft-versus-myeloma eradication of the malignant
clone by immunocompetent donor lymphocytes,
graft-versus-host disease (GVHD), and high rates of
treatment-related mortality plague, this modality and
use remains controversial [4,5].
Relapse rates after AHCT are extremely high
compared with allogeneic hematopoietic cell trans-
plantation. These results reflect, in part, lack of
a graft-versus-myeloma effect and reinfusion of autol-
ogous tumor cells [6]. A number of investigators have
proposed that the induction of GVHD and attendant
graft-versus-tumor effect may reduce relapse rates
and improve overall patient outcome in a variety of
malignancies [7-13].
Herein we report 9 cases of spontaneous autolo-
gous GVHD in plasma cell myeloma patients undergo-
ing initial AHCT. We compared 6 of these 9 patients
with 42 concurrently treated patients who did not de-
velop this condition with respect to age, gender, stage
at diagnosis, treatment type, CD34
1
cell dose, pres-
ence of diarr heal illness during the peritransplant pe-
riod, and time to engraftment. Subsequently, we
retrospectively analyzed all 9 patients hematopoietic
progenitor cell grafts using enzymatic amplification
staining (EAS) enhanced flow cytometry to evaluate ex-
pression of various transcription regulators, pathway
molecules, and surface receptors compared with 42
unaffected controls.
PATIENTS AND METHODS
Between March 2006 to September 2009, 48 pa-
tients underwent AHCT for plasma cell myeloma at
University Hospitals Case Medical Center, Cleveland,
Ohio. The institutional review board for human inves-
tigation granted approval for this retrospective study.
Three additional patients with the clinical syndrome
were added after the aforementioned time period for
flow cytometric analysis, but were not included in
the case-contr ol analysis. All data were obtained via
retrospective chart reviews using the electronic medi-
cal record and paper charts. Myeloma staging was re-
ported using the Durie-Salmon [14] system. Blood
cells were mobilized from patients who were treated
uniformly with cyclophosphamide 4 g/m
2
intravenous,
filgrastim (Amgen, Thousand Oaks, CA) 10 mg/kg
once or twice daily subcutaneous (determined by
resting-state the blood CD3 4
1
cell concentration),
and prednisone 2 mg/kg/day by mouth for 4 days.
Apheresis was undertaken when the blood CD34
1
cell
count exceeded 10/mL. Filgrastim was continued until
the last day of apheresis. A multilumen central venous
apheresis catheter was placed either in the internal jug-
ular or subclavian vein for blood cell mobilization and
subsequent transplantation. The date of transplant was
defined as day T 5 0, and all days were calculated from
this metric. The conditioning regimen consisted of
amifostine 740 mg/m
2
i.v. on days T22 and T21
and melphalan 200 mg/m
2
i.v. on day T21. All pa-
tients received filgastrim support from day T11 until
3 consecutive days after bloo d neutrophil count
.500/mL. Quantification of amounts and days of diar-
rhea could not be obtained in all patients because of in-
adequacies of documentation. Autologous GVHD was
treated at the discretion of the attending physician
using: corticosteroids (methylprednisolone or predni-
sone 1-2 mg/kg/day); budesonide 3 mg by mouth 3
times daily; or the combination. Subse quent weaning
was undertaken at the discretion of the treating physi-
cian without use of a predetermined protocol.
Statistical Plan and Analysis
After recognizing 6 cases with autologous GVHD,
we conducted a case-control study using 42 plasma cell
myeloma patients who underwent an AHCT identi-
cally managed, yet who did not develop autologous
GVHD during the same pe riod (controls). The com-
parison between cases and controls was performed us-
ing the chi-square test or Fisher exact test for categoric
variables and using the Student t test for comparing
continuous measurements. The association between
2 continuous variables was estimated using the Pear-
son correlation coefficient. The cumulative neutrophil
recovery rate was estimated using the Kaplan-Meier
method, and the difference between cases and controls
was tested using the log-rank test [15]. All tests were 2
sided and P \ .05 was considered significant.
Flow Cytometric Analysis
Aliquots of peripheral blood mononuclear cells,
cryopreserved at the time of initial hematopoietic
cell mobilization, were available for the 9 affected pa-
tients and 42 controls. Cryopreserved samples were
thawed and analyzed via EAS, which provides 10- to
100-fold greater sensitivity than standard staining
procedures. This technology has been demonstrated
to be precise, reproducible, quantitative, and stable
upon freezing/thawing [16-20]. EAS obtains
significant amplification by catalyzing the deposition
of tyramide on the analyte-expressing cell. CD34
1
cells in the samples were assessed for expression of
(1) pathway molecules (PTEN, pAkt[308], pAkt
[473], b-catenin, GAB2); (2) transcription regulators
(HoxB4, BMI-1, GATA-2, c-MYC, E47, RUNX1);
and (3) surface receptors (IL-23R, IL-3R, CXCR4,
CD117, CD130). The peripheral blood mononuclear
cells were stained by Pathfinder Biotech (Clevela nd,
OH). Briefly, antibodies specific for CD34 and conju-
gated with AlexaFluor 647 were used to identify
hematopoietic progenitor cells. The expression levels
Biol Blood Marrow Transplant 17:970-978, 2011 971Autologous GVHD in Myeloma
Page 2
of PTEN, phospho-Akt (thr308), phospho-Akt
(ser473), b-catenin, HoxB4, RUNX1, Bmi-1,
GATA-2, E47, and GAB2 were assessed after fixation
and permeabilization by processing the signals in the
fluorescein channel with EAS. The expression levels
of IL-23R, CD123, CD117, CD130, and CXCR4
were assessed on live cells by processing the signals
in the fluorescein channel with EAS. Stained samples
were analyzed on a FACSCalibur flow cytometer
and CELLQuest software. Isotype/subtype matched
immunoglobulins were used to control the expression
levels of the various analytes. The median fluorescence
ratio was obtained from the median fluorescence in-
tensities for the specific antibodies versus matched
control immunoglobulin.
Antibodies
Rabbit antibodies specific for PTEN, phospho-
Akt (thr308), phospho-Akt (ser473), and b-catenin
were purchased from Cell Signaling Technology
(Danvers, MA). Rabbit antibodies specific for
HoxB4 and RUNX1 were purchased from Epitomics
(Burlingame, CA). Antibodies specific for Bmi-1,
GATA-2, IL-23R, and CXCR4 were purchased from
R&D Systems (Minneapolis, MN). Antibodies specific
for E47 and CD123 were purchased from BD Biosci-
ences (San Jose, CA). Antibodies specific for GAB2
and CD130 were purchased from abcam (Cambridge,
MA). Antibodies specific for CD117 and CD34
were purchased from BioLegend (San Diego, CA).
Antibody specific for c-MYC was purchased from
Invitrogen (Seattle, WA).
RESULTS
Table 1 shows the patient-, disease-, treatment-,
and symptom-related information for the 5 women
and 4 men median (range) age 62 (38-68) years. Autol-
ogous GVHD was confirmed by histologic examina-
tion in all 9 patients. This phenomenon occurred at
a median (range) time to onset of symptoms at 8
(2-27) days after initial AHCT. Biopsies were obtained
at a median (range) of 14 (11-45) days after transplant.
All symptoms responded promptly to therapy with no
long-term sequelae. Three of the 9 patients have
relapsed or died.
Case-Control Analysis
Six patients affected in the predetermined time pe-
riod were included in a case-control analysis; the 3 addi-
tional affected patients were not included in this
analysis. During the same time period, 42 plasma cell
myeloma patients (controls) underwent an AHCT and
were compared with the 6 affected patients (Table 2).
Eleven of the patients (26%) in the unaffected group
reportedly had a diarrheal illness compared with 100%
Table 1. Nine Patients Affected with Autologous GVHD
Age/Gender Initial Stage*/Type Prior Therapies
CD34
+
10
6
/kg
Cell Dose
Symptom
Onset after
HCT (Days)
Time to
Biopsy after
HCT (Days)
Sites/Stage
aGVHD GVHD Therapy
Time To
Neutrophil
Recovery (Days)
55 years/M IIIB IgG-k Bort/Dex; Cy 16.4 7 14 GI 3 Methylpred 1 mg/kg and Bud 8
62 years/F IIIA IgG-k Thal/Dex;Bort/Dex;Mel/Pred 8.1 27 45 GI 4 Methylpred 1 mg/kg and Bud 11
53 years/F IIIB IgG-k Thal/Dex 5.2 12 19 GI 3/liver 2 Methylpred 2 mg/kg 12
38 years/F IIA IgG-k Thal/Dex/Bort 5.3 9 14 GI 3/liver 2 Pred 2 mg/kg 12
67 years/F IIIA IgA-l Len/Dex 15.7 8 21 GI 3 Pred 1 mg/kg 10
56 years/M IIIA IgA-k Len/Dex 15.0 8 11 GI 3/skin 2 Pred 2 mg/kg and Bud 10
64 years/F IIIB Free-k CyVD;Bort/Dox;Len/Dex 6.3 2 15 GI 3 Methylpred 1 mg/kg 11
66 years/M† IA IgA-k Len/Dex;Bort/Dox 23.3 10 12 GI 3 Methylpred 2 mg/kg 10
68 years/M† IIIA IgG-k Vel/Len/Dex 8.7 3 12 GI 2 Pred 1 mg/kg 10
HCT indicates hematopoietic cell transplantation; aGVHD, acute graft-versus-host disease; GVHD, graft-versus-host disease; Bort, Bortezomib; Dex, Dexamethasone; Cy, Cyclophosphamide; Methylpred, Methyl-
prednisolone; Bud, Budesonide; Thal, Thalidomide; Pred, Prednisone; Len, Lenolidamide; Dox, liposomal doxorubicin.
*Stages reported as per Durie-Salmon [14].
†These 3 patients were added to the original 6 affected patients and were not included in the case-control analysis (Table 2). Their hematopoietic progenitor cells were analyzed in the flow cytometric analysis.
972 Biol Blood Marrow Transplant 17:970-978, 2011H. M. Lazarus et al.
Page 3
for the affected cases (P 5 .001). Sigmoidoscopy or bi-
opsieswere not obtained in these unaffected patients be-
cause of other clinical diagnoses. Indeed, 2 evaluations
revealed Clostridium difficile enterocolitis; the other pa-
tients were suspected of having antibiotic-associated di-
arrhea or melphalan-induced mucositis that resolved
without sequelae or extended duration. Age, CD34
1
cell dose infused, time to neutrophil recovery, and num-
ber of regimens and types of therapy were similar for
both groups. With a limited number of cases, there
were trends but not of statistical significance: Patients
affected with autologous GVHD were more likely to
be female (67% vs. 36%, P 5 .197)
and more had advanced-staged disease (83% vs. 48%,
P 5 .191).
Flow Cytometric Analysis
Coincident with this investigation, we were inde-
pendently investigating molecular expression levels
in CD34
1
hematopoietic progenitor cells from the
same patients. Because the onset of autologous
GVHD was early after transplantation, we considered
the possibility that there are differences in the cellular
inocula between samples that resulted in autologous
GVHD and those that did not. Consequently, we an-
alyzed the molecular expression resul ts from the 9
clinically affected patients compared with controls,
with the idea that molecular expression levels in the
CD34
1
cells may relate to molecular expression levels
in T cells or relevant T cell subpopulations (Table 3).
Three targets, GATA-2, CD130, and CXCR4, had
statistically significant increased expression (P \ .05)
compared with controls. A fourth target (c-MYC)
was marginally sign ificant (P 5 .073). Representative
results are shown (Figure 1). There was no correlation
between expression of CXCR4 and CD130, CXCR4
and c-MYC, CXCR4 and GATA-2, CD130 and
c-MYC, or CD130 and GATA-2. GATA-2 and
c-MYC positively correlated in CD34
1
cells from
patients affected with autologous GVHD, with r 5
.67 (P 5 .048) compared with control patients
(Figure 2).
DISCUSSION
More than 20 years ago, Hood and colleagues [21] de-
scribed an autoimmune syndrome similar to acute
GVHD after AHCT termed ‘ ‘autoaggression’ syn-
drome, or autologous GVHD. This c ondition tended
to be milder than classic GVHD after allogeneic trans-
plantation, most commonly involved the skin (rarely the
gastrointestinal tract or liver), infrequently required ther-
apy, and often was self-limited [7,21-24]. Subsequently,
autologous GVHD uncommonly has been reported to
occur spontaneously and can be induced by design after
administration of posttransplant immune modulation
with cyclosporine A, interferon-g, or the comb ination ,
in an attempt to stimulate a graft-versus-tumor response
[8-13, 21, 24] .
We report herein 9 plasma cell myeloma patients
who appeared to develop the syndrome of autologous
GVHD spontaneously at a median (range) time to on-
set of symptoms at 8 (2-27) days after initial AHCT.
Forty-two similarly treated patients during this 2-
year period did not exhibit symptoms to warrant clin-
ical evaluation, and we found no clinical differences to
be able prospectiv ely to identify patients at highest
risk. In contrast to previous autologous GVHD re-
ports, only 1 patient had clinical skin involvement,
whereas in all others the gastrointestinal (GI) tract
findings predominated, and 2 subjects had liver dys-
function as well. Ideal ly, it would have been useful to
compare diarrheal output and incidence between af-
fected and unaffected patients to identify this syn-
drome on clinical grounds; the retrospective nature
of this analysis, however, precluded this undertaking.
Table 2. Comparison of Clinical Factors
Variable Unaffected (n 5 42) Autologous GVHD (n 5 6) P value
Median (range) age (years) 55 (41-69) 55.5 (38-67) .954
Gender (male/female) 27/15 2/4 .197
Stage (Durie-Salmon) .191
I/II 22 (52%) 1 (17%)
III 20 (48%) 5 (83%)
CD34
+
cell dose (10
6
/kg): mean (range) 8.82 (3.51-17.5) 10.9 (5.2-16.4) .269
Pretransplant therapies
Thalidomide 25/42 (60%) 3/6 (50%) .683
Melphalan 2/42 (5%) 1/6 (17%) .336
Lenalidomide 20/42 (48%) 2/6 (33%) .674
Bortezomib 10/42 (24%) 3/6 (50%) .323
Doxorubicin 7/42 (17%) 0/6 (0%) .573
Cyclophosphamide 1/42 (2%) 1/6 (17%) .237
Multiple lines therapy 12/42 (29%) 2/6 (33%) .99
Neutrophil recovery (days): median (range) 11 (9-26) 10.5 (8-12) .357
Tandem autologous transplant 11/42 (26%) 0/6 (0%) .313
Discharge summary reported diarrheal illness 11/42 (26%) 6/6 (100%) .001
GVHD indicates graft-versus-host disease.
Biol Blood Marrow Transplant 17:970-978, 2011
973Autologous GVHD in Myeloma
Page 4
These features are most consistent with the auto-
logous GVHD syndrome; engraftment syndrome is
an unlikely possibility [25-27]. Signs and symptoms
in our patients were confined mainly to the GI tract.
Only 1 patient had a rash and 3 had a fever at time
of biopsy, features considered the most salient
diagnostic aspects of engraftment syndrome.
Additionally, affected patients did not exhibit the
findings of capillary leak, marked weight gain,
noncardiogenic pulmonary edema, and multiple
organ dysfunction syndrome.
Further, we examined a number of molecular tar-
gets in the infused hematopoietic progenitor graft.
We identified CD130 [28,29], GATA-2 [30-32],
CXCR4 [33-35], and c-MYC [36,37], molecules
well-described as integral in hematopoietic cell devel-
opment, as having increased expression in CD34
1
he-
matopoietic progenitor cells in those patients affected
with autologous GVHD.
Our patients were treated with bortezomib. CD130
[38,39] signaling is perturbed under the influence of
bortezomib. Those patients unaffected by autologous
GVHD (controls) did not exhibit these findings in
their autologous grafts. Overexpression of these 4
markers has not been implicated previously in
autologous GVHD. Although we did not specifically
evaluate T cells, we postulate that exposure over time
to the various immunomodulating therapies used for
induction treatment affected not only the CD34
1
cells but also T cells or relevant T cell subpopulations
capable of mediating GVHD. After infusion, these
affected hematopoietic progenitor cells then
encounter a host that has been further altered by the
high-dose melphalan preparative regimen; such a situa-
tion leads to autologous GVHD. Further prospective
study is needed to validate these findings.
This phenomenon in our case series appears to be
more common and with features different than most
previously reported cases of spontaneously occurring
autologous GVHD. Only 1 of our patients appeared
to have skin involvement, although we did not perform
random cutaneous biopsies in unaffected skin and can-
not exclude subclinical manifestations. Further, he-
patic evidence of autologous GVHD, an extremely
uncommon finding, apparently affected 2 AHCT re-
cipients. Onset in our patient population was much
earlier than in any of the other series.
Kline and colleagues [40] reviewed reports of spon-
taneously occurring autologous GVHD. This syn-
drome developed in a variety of disorders after
AHCT, but the incidence was uncommon. Most af-
fected patients had skin involvement, but GVHD
Table 3. Correlations between Autologous GVHD and Molecular Expression in Hematopoietic Progenitor Cells
Autologous GVHD Mean (Standard Deviation) N P
PTEN 2 13.0 (8.8) 38 .518
+ 15.2 (2.5) 8
Phospho-Akt 2 8.8 (1.0) 32 .329
(thr308) + 11.1 (1.2) 6
Phospho-Akt 2 3.0 (0.3) 36 .285
(ser473) + 2.5 (0.3) 9
b-catenin 2 6.8 (0.6) 34 .402
+ 5.7 (0.9) 9
GAB2 2 2.6 (0.3) 20
8
.579
+ 3.1 (087)
HoxB4 2 6.0 (0.7) 38 .744
+ 5.6 (0.6) 9
Bmi-1 2 14.7 (2.0) 38 .377
+ 16.4 (2.2) 9
GATA-2 2 37.5 (4.9) 38 .005
+ 69.1 (6.7) 9
c-MYC 2 50.9 (7.1) 30 .073
+ 80.0 (16.5) 9
E47 2 27.9 (3.0) 26 .609
+ 31.3 (7.2) 9
RUNX1 2 9.8 (0.8) 20 .412
+ 8.5 (1.4) 9
IL-23R 2 33.9 (4.7) 27 .654
+ 38.4 (10.2) 9
IL-3R 2 12.0 (1.8) 38 .166
+ 8.5 (1.6) 9
CXCR4 2 5.4 (0.7) 38 <.0001
+ 1.9 (0.3) 9
CD117 (c-Kit) 2 34.9 (5.4) 37 .853
+ 32.8 (6.5) 9
CD130 2 6.2 (0.7) 36 .02
+ 9.8 (1.5) 9
GVHD indicates graft-versus-host disease.
974 Biol Blood Marrow Transplant 17:970-978, 2011H. M. Lazarus et al.
Page 5
affecting the GI tract was rare. Time to clinical onset in
several series ranged from 11 to 37 days after AHCT.
Holmberg and colleagues [22] noted that 90 of 681
(13%) AHCT recipients, many of whom received the
GI-toxic regimen ‘BUMELT’ (busulfan/m elphalan/
thioTEPA), developed autologous GVHD. Findings
included persistent nausea, vomiting, and diarrhea,
mucosal abnormalities at endoscopy, and histology
showing apoptotic crypt cells with or without lym-
phoid infiltrates. They surmised that the resultant GI
mucosal damage increases the risk of autologous GI
GVHD as activated cytotoxic T cells ‘home’ to and
cause further GI mucosal damage. Women, especially
those with the diagnosis of breast cancer, had a 3-fold
higher rate of developing this syndrome compared
with men. Mean time to establishing a histologically
proven diagnosis and starting prednisone treatment
was 42 and 45 days, respectively, after AHCT. Most pa-
tients responded promptly to corticosteroid therapy. It
should be noted that 20% of affected patients were
treated with interleukin-2.
Recently, Drobyski and colleagues [41] described 5
cases of severe GI tract autologous GVHD in a popu-
lation of 250 plasma cell myeloma AHCT recipients.
The syndrome was corticosteroid-refractory in all 5
and was fatal in 4 of the affected patients. In contrast
to our patients, autologous GVHD tended to develop
after the second (tandem) autograft and was much later
in onset. Only 2 of the 5 affected patients received
therapy with thalidomide, lenalidomide, or bortezo-
mib; the remaining patients received vincristine, adria-
mycin, and dexamethasone. The T and B cell subsets
in the hematopoietic cell grafts did not differ between
affected and the unaffected patients. They speculated
that the second AHCT might compromise endoge-
nous peripheral regulatory mechanisms and predis-
pose these patients to autoimmunity. Cer tainly, in
comparison to Drobyski and colleagues’ [41] report,
our population lacked the severe morbidity and mor-
tality.
We hypothesize several reasons for these findings
including perturbation of the immune system
Figure 1. Representative flow cytometric analysis of CD34
1
cells in mobilized blood samples. Mononuclear cells from 4 different mobilized blood
samples were stained for CD34 expression (upper row). The cells were also stained with control immunoglobulin (middle row) or with specific anti-
bodies (lower row) and processed by EAS for high-resolution immunophenotyping. The specific antibodies shown are CXCR4 (lower left), CD117
(lower middle left), GATA-2 (lower middle right), and Bmi1 (lower right). The stains for CXCR4 and CD117 are surface stains without fixation and
permeabilization. The stains for GATA-2 and Bmi-1 are intracellular stains with fixation and permeabilization. The amplified signals (middle and lower
rows) are shown only for the CD34
1
gates (upper row). Representative results are shown from 4 different donors in order to demonstrate the con-
sistency in CD34
1
cell delineation.
Biol Blood Marrow Transplant 17:970-978, 2011
975Autologous GVHD in Myeloma
Page 6
composition because of the underlying disease, the use
of new immunomodulating medications, and the con-
ditioning regimen for the AHCT. Frassanito and col-
leagues [42] demonstrated that CD8
1
/CD57
1
lymphocytes contribute to the immune dysregulation
in plasma cell myeloma. Many investigators have de-
scribed the perturbations in the immune system that
occur in response to myeloma therapies, especially
the new immunomodulating therapies. The antitumor
properties of thalidomide and its analogues are incom-
pletely understood but likely include antiangiogenic,
antiproliferative, and anti-inflammatory effects, as
well as immunomodulation of T cells and NK cells.
Galustian and colleagues [43] demonstrated that lenali-
domide and pomalidomide inhibit T regulatory cell ex-
pression and suppressor activity against self-reactive
cells in vitro. Also, thymic function in adults receiving
high-dose cytotoxic therapy is poor, and patients are
susceptible to attack by autoreactive immune cells.
Two groups [44,45] have demonstrated in separate
reports that high-dose melphalan significantly sup-
presses the overall number and function of T cells.
Melphalan is an agent used commonly in condi-
tioning plasma cell myeloma patients for AHCT that
frequently leads to profound mucositis and symptoms
akin to GVHD of the GI tract. This overlap could lead
to delay in diagnosing autologous GVHD and institut-
ing appropriate therapy. We believe it important to
follow myeloma patients who develop autologous
GVHD to know if they expe rienced differences in re-
lapse and survival rates compared with patients who
did not develop autologous GVHD. To date, autolo-
gous GVHD has not been associated with an enhanced
graft-versus-tumor effect [40]. In contrast to chronic
GVHD, autologous GVHD in the allogeneic
transplant setting is not reportedly associated with
a decreased relapse rate. Indeed, in our initial cohort,
3 of 9 patients had disease progression.
This uncommon complication of autologous
GVHD in plasma cell myeloma AHCT recipients
needs to be recognized early, as it appears to be treat-
able. This syndrome may be underreported as 1 of the
many possible causes of diarrhea in this population.
These findings easily can be mistaken for melphalan-
induced mucositis or antibiotic-associated colitis.
Therefore, patients who develop intractable symptoms
and signs of GI tract injury, with or without hepatic
dysfunction, should undergo appropriate investiga-
tions that may include endoscopy and possibly muco-
sal biopsy in order to start GVHD therapy promptly.
Expression of CD130, GATA-2, and CXCR4 is al-
tered in the hematopoietic progenitor cells of patients
affected with autologous GHVD. We hypothesize that
the differences we observed in the hematopoietic pro-
genitor cells are associated with differential molecular
expression levels in T cells or in relevant T cell sub-
populations. Further prospective study is needed to
validate these markers to see if a model could be de vel-
oped to predict this syndrom e.
ACKNOWLEDGMENTS
Financial disclosure: The authors have no conflict of
interest.
AUTHORSHIP STATEMENT
H.M. Lazarus designed research, performed re-
search, collected data, analyzed and interpreted data,
Not associated with aGVHD Associated with aGVHD
r = 0.3 (p = 0.114) r = 0.67 (p = 0.048)
Figure 2. Correlation of cMyc and GATA-2 expression levels in CD34
1
cells. CD34
1
cells from various samples of mobilized blood from patients with
multiple myeloma were stained for the expression of c-MYC and separately for the expression of GATA-2. The cells were processed by EAS for high-
resolution immunophenotyping. Samples not associated with the subsequent development of autologous GVHD in the transplanted patients (left panel )
and samples that were associated with the subsequent development of autologous GVHD in the transplanted patients (right panel) are shown. The
distributions of median fluorescence ratios are shown for the various samples. Coefficients of correlation and P values are shown. Abbreviations:
aGVHD indicates acute graft-versus-host disease.
976 Biol Blood Marrow Transplant 17:970-978, 2011H. M. Lazarus et al.
Page 7
and wrote the manuscript. S.R. Sommers performed re-
search, collecteddata, analyzedandinterpreted data, and
wrote the manuscript. L.M. Arfons analyzed and inter-
preted data. P. Fu analyzed and interpreted data, and
performed statistical analysis. S.A. Ataergin collected,
analyzedandinterpreted data,and wrote the manuscript.
N.M. Kaye contributed vital new reagents or analytical
tools. F. Liu contributed vital new reagents or analytical
tools. T.L. Kindwall-Keller analyzed and interpreted
data. B.W. Cooper analyzed and interpreted data. M.J.
Laughlin analyzed and interpreted data. R.J. Creger an-
alyzed and interpreted data. P.M. Barr analyzed and in-
terpreted data. S.L. Gerson analyzed and interpreted
data. D. Kaplan designed research, performed research,
analyzedandinterpreted data,wrote the manuscript, and
contributed vital new reagents or analytical tools.
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