Analysis of &Globin Mutations Shows Stable Mixed

Transcription

Analysis of &Globin Mutations Shows Stable Mixed
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Analysis of &Globin Mutations Shows Stable Mixed Chimerism in Patients
With Thalassemia After Bone Marrow Transplantation
By J. Kapelushnik, R. Or, D. Filon, A. Nagler, G. Cividalli, M. Aker, E. Naparstek,
S.Savin, and A. Oppenheim
The seventh patient hasless than 10%donorcells with,
/?-thalassemia major(TM) is causedby any of approximately
surprisingly, onlyminimal transfusion requirements. The de150 mutations within the /?-globin gene. To establish the
tection of /?-globin gene point mutation,as used here, is a
degreeofchimerism
afterbonemarrowtransplantation
highly specific and sensitive marker for engraftment MC
and
(BMT), we have performed molecular analysis of P-globin
in patients with thalassemia. In light of its specificity, the
mutations in14 patients with TM over a periodof 10 years.
All patients underwent T cell-depleted allogeneic BMT from method is applicable in all cases ofTM, as it is independent
of sex and othernon-globin-related DNAmarkers. The high
HLA-identicalrelated donors,usingeither
in vitroT-cell
a
incidence of MC found inour patients may be consequence
depletion with CAMPATH 1M andcomplementor in vivo
MC was associated
depletion using CAMPATH 1Gthe
in bone marrow collection of the pre-BMT T-cell depletion. Because
with transfusion independence, complete eradication of rebag. To date, at different time periods after BMT, seven pasidual host cells for effective treatment of TM and possibly
six of these patients,
tients have some degree of chimerism;
other genetic diseases may prove not to be essential.
all blood transfusion-independent, have donor cells in the
0 7995 by The American Society of Hematology.
range of 70% to 95%. with stable mixed chimerism (MC).
H
IGH-DOSE CHEMOTHERAPY followed by allogeneic bone marrow transplantation (BMT) is the treatment of choice for a wide range of nonmalignant hematologic diseases, including &thalassemia major ( T M > . l 2 * The
pretransplant chemotherapy has a dual purpose: to kill hematopoietic host cells so as to gain bone marrow space and, at
the same time, to induce adequate immunosuppression to
prevent rejection of donor stem cells.
Despite the significant and continuing advances in conditioning regimens, patients may ultimately develop either persistent host lymphohematopoietic cells or relapse with the
basic disease. A number of studies have shown that patients
in good clinical condition post-BMT and presenting with
normal hematologic parameters may still harbor minimal
amounts of residual host hematopoietic cells in their bone
marrow and peripheral blood, as determined by highly sensitive technique^.^.^ The methods used thus far to detect chimerism after BMT for nonmalignant disorders include detection
of sex chromosomes in cases of sex disparity between donor
and recipient: analysis of hypervariable regions of the human genome by polymerase chain reaction (PCR) amplificat i ~ n , * . and
~ - ' ~AB0 typing."
The present work focuses on the detection of point mutations in the &globin gene of patients with TM undergoing
BMT. This innovative technique aIlows us to establish the
degree of chimerism by measuring the level of residual host
P-globin genes, using the point mutation as a marker, and
permits correlation between the degree of chimerism and the
clinical outcome after BMT.
MATERIALS AND METHODS
Patients. The study comprises 14 informative patients with TM
(9 males and five females; age range, 1 to 7 years; median, 4 years
at presentation) treated during the last decade at the Department of
Bone Marrow Transplantation of the Hadassah University Hospital,
Jerusalem, Israel. All patients received allogeneic BMT from HLAidentical, related donors. Six pairs were sex-mismatched.
Pretransplant conditioning. All patients received oral busulfan
(4 mgflrg/d X 4 days) and intravenous (IV) cyclophosphamide (50
mg/kg/d X 4 days). Thiotepa IV (5 m@g X 1 day) was added for
four patients. Total lymphoid irradiation (TLI), 1O
, OO cGy given in
five daily fractions, was performed in 10 patients, and four patients
received IV rat anti-human CDWS2 (IgG 2b) antibody (CAMPATH
Blood, Vol86, No 8 (October 151, 1995: pp 3241-3246
1G) (0.2 mg/kg/d X 4 days) for in vivo depletion of hostlymphocytes
before chemotherapy.
Graft-versus-host disease (GVHD)prophylaxis. All patients received T cell-depleted bone marrow achieved by treatment either in
vitro (nine patients) with CAMPATH IM, with donor serum as
source of complement CAMPATH, or in vivo with CAMPATH 1G
directly in thebone marrow collection bag (five patients). In the latter
case, depletion was most likely achieved by antibody-dependent cellmediated cytotoxicity. CAMPATH 1Mand CAMPATH 1G were
provided by Drs G . Hale and H. Waldmann (Department ofPathology, Dunn School of Pathology, Oxford, UK). The use of
CAMPATH 1M in vitro and CAMPATH 1G in the marrow collecting bag has been described previ~usly.'~"~
Rejection prevention. Cyclosporin A (3 mg/kg/d)was administered intravenously to all patients from day -1 until engraftment
(polymorphonuclear cells, 2750 mmz).
DNA preparation and PCR. DNA was prepared from peripheral
blood according to standard procedures.16PCR was performed with
Taq polymerase (Appligene, Strasbourg, France) using the conditions recommended by the manufacturer. The following primers,
spanning the first and second exons of the 8-globin gene from 166
nucleotides (nt) upstream of the cap site to 138 nt in the IVS2 site,
were used: PS (S' primer), CCAACTCCTAAGCCAGTGCC; and
P12 (3' primer), CTGAGACTTCCACACTGATGC.The amplification cycle consisted of 1 minute at 92"C, 1.5 minutes at 62"C, and
1.5 minutes at 7 2 T , giving an amplification product of 799 bp.
Testingfor mutant alleles. Before BMT, the B-thalassemia mutation(s) of the patient was identified: in parallel, the @-globingenotype of the donor was determined. Some of the donors were normal,
while others were thalassemia carriers. After the transplantation,
DNA samples obtained from peripheral blood were tested for chimeFrom the Departments of Pediatrics, Hematology, and Bone Marrow Transplantation, Hadassah University Hospital, Ein Kerem, Jerusalem, Israel.
Submitted March 17, 1995; accepted June 19, 1995.
Supported in part by a grant from the Social Security Institution
in Israel and by the Robert A. Rosenblum Research Fund.
Address reprint requests to Reuven Or, MD, Department of Bone
Marrow Transplantation, Hadassah University Hospital, Jerusalem
91 120, Israel.
The publication costs ofthis article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. seetion 1734 soleIy to
indicate this fact.
0 1995 by The American Society of Hematology.
oooS-4971/95/8608-04$3.00/0
3241
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KAPELUSHNIK ET AL
3242
A
PCR cycles
15
20
18
23
25
30
B
z
a
U
10’
Fig 1. Kinetics of the p-globin PCR. The reaction
was set up withnormal control DNA in a total of 300
p L that was divided equally into six tubes. Tubes
were withdrawn after different numbers of amplification cycles, as shown in panel A. Aliquots of the
PCR products (10 pL) were analyzed by Southern
blotting (B). For quantification, the membrane was
cut, and the bands were counted in a scintillation
counter.
1
10
15
20
25
30
Cycles
rism as follows: equal aliquots of the PCR products (5 to 15 pL) were
denatured in 0.4 molL NaOH125 mmolL EDTA for 20 minutes on
ice and spotted onto two duplicate nylon membranes together with
appropriate controls, namely, donor DNA controlandcontrolsfor
each 8-globin mutation (homozygotes, heterozygotes, and normal).
Hybridization was performed with excess allele-specific oligonucleotide probes (1 to 2 ng) labeled with [.‘’P]7-adenosine triphosphate
(ATP).” For each patient, one membrane was hybridized with the
mutantprobeandthe
second, withthe respective normalprobe.
Quantification of the autoradiograms was performed as follows. Until 1993, radioactive counts weredeterminedbycuttingthe
spots
from the membrane and measuring in a scintillation counter, since
1993, theradioactivity of the spots ismeasureddirectly
using a
phosphorimage analyzer.
RESULTS
The first objective was to establish the sensitivity and
reliability of the method. For dependable quantification of
the ratio of normal to mutant alleles, measurements must be
performed within the linear range of detection. The PCR is
expected to start at an exponential rate and to remain so
until one of the reaction components becomes limiting. We,
therefore, examined first the progress of the PCR under our
conditions. Because the reaction was exponential up to 25
cycles (Fig l ) , 23 cycles were used for all subsequent tests.
Linearity of the signal obtained by hybridization depends
on the ratio between DNA and probe. The PCR products
35
were spotted onto duplicate membranes at different DNA
concentrations; membranes were then hybridized to 1 ng/
mL (Fig 2B, closed circles) and 3 ng/mL probes (Fig 2B,
open circles). Atboth probe concentrations, the reactions
were linear over a wide range.
Measurement of radioactivity by phosphorimage analysis
showed quantification of the results to be highly reproducible. Replicate analyses of a sample, at the same or different
DNA concentrations, demonstrated an experimental error of
S 1%. Repeated analyses of the same sample at intervals of
months were likewise reproducible. We, therefore, consider
a 2% hybridization signal as significant. Hence, the method
can detect 2% donor (or 2% residual host) cells when both
donor and recipient are homozygous, or establish 4% donor
cells when the donor is heterozygous (or the recipient is
compound heterozygous).
After BMT, peripheral blood samples were analyzed sequentially for detection of the P-globin gene point mutations
for all14 patients. Table 1 lists the clinical parameters of
the 14 patients and the outcome of their BMTs: Five patients
(43%) were found to have full engraftment (lOO% donor
cells) based on analysis of the P-globin alleles. These five
patients are in excellent clinical condition, exhibiting normal
growth and development; their hemoglobin levels are comparable with those of the respective donors. In seven patients
(50%), mixed chimerism (MC) was detected by the P-globin
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D-GLOBIN MUTATION IN THALESSEMIA POST-BMT
3243
A
Relative
DNA concentration
B
lo5
10'
E
P
v
Fig 2. Linear range of DNA concentration for allele-specific detection. /.?-globinPCR productsof normal control DNA were serially diluted (twofold each
time) and blotted onto duplicate nylon membranes.
The blots were hybridized to a '2P-labeled oligonucleotide probe. (A) Autoradiogram of hybridization
to 1 nglmL probe. (B)Quantitative analysis: the
membraneswere cut, and the spots were counted in
a scintillation counter. Hybridizationwas performed
with 1 nglml ( 0 )and 3 ng/rnL (0)
probes.
lo3
.03 .06 . l 2 2 5 .S
1
:c"
I
0
lo2
0
10'
gene point mutation method (and not by any of
the other
engraftment markers); six of these patients have 73% to
96% donor cells with stable MC, are independent of blood
transfusions, and are in excellent clinical condition. The seventh patient has 4% to 7% donor cells with only a minimal
transfusion requirement; ie, she presents clinically with thalassemia intermedia. The remaining 2 patients (7%) have no
detectable donor genes (ie, 100% host cells) and suffer from
TM. None of the 14 patients showed signs of acute or chronic
GVHD. To date, the period of follow up of the entire group
of patients ranges between 6 months and 11 years (median,
4 years).
In the seven patients withMC, only one was sex-mismatched: a male was transplanted from a female donor (UPN
338, Table 1). For this recipient, PCR for the detection of
Y chromosome was positive. The other five sex-mismatched
recipients did not have MC.
In the seven patients with MC,the degree of chimerism, as
expressed by the host-to-donor DNA ratio, remains relatively
stable post-BMT (follow up of 3 to 68 months; median, 18
months). Despite the presence of MC, the six patients with
greater than 70% donor cells have redblood cell counts
similar to those of their donors; the stable MC in this group
of patients, therefore, indicates sustained engraftment and
certainly not recurrence of the basic disease.
Thus far, no correlation was found to exist between the
01
I
I
.l
1
Relative
DSA
10
concentration
pretransplant conditioning regimen or mode of T-cell depletion (TCD) and the incidence of chimerism.
DISCUSSION
In this study, we present the detection of-the P-globin
gene point mutation as an innovative method to confirm
engraftment, detect minimal residual disease, and establish
degree of chimerism in patients with thalassemia after BMT.
Whereas other methods depend on neutral, nonrelated markers for the evaluation of chimerism, the strategy described
here is based on the direct measurement of the diseasecausing gene, albeit in irrelevant cell lineages.
BMT for thalassemia is usually performed with stem cells
from closely related, HLA-matched donors, prohibiting the
use of HLA as a marker. The AB0 antigens as engraftment
markers are of value only in cases of A B 0 mismatch and,
furthermore, have no significance with respect to minimal
residual disease. DNA-based gender determination is limited
to situations of donor-host sex disparity. The specificity of
the 0-globin gene point mutation test, on the other hand,
makes it applicable for every patient with TM receiving
BMT.
The method, based on allele-specific oligonucleotide hybridization, is highly sensitive and capable of detecting an
allele even at a level as low as 1%. Thus, in the case of a
normal donor, even 1% to 2%residual host cells are detected.
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3244
KAPELUSHNIK ET AL
Table 1. Chimerism and Outcome of BMT
Chimerism
UPN
Age at
BMT
(yrs)
P-Globin Genotype
Conditioning
RegimenflCD
Patient
Donor
50
152
170
184
1
2
1
1
BuCy, TLI/lM
BuCy, TLI/lM
BuCy, TLI/l M
BuCy, TLI/l M
FS44/FS5
IVS1, l/IVSl, 1
IVS2, l/lVS2, 1
IVS1, llO/IVSl, 110
FS44/A
IVS1, l / A
IVS2, 1/A
IVS1, 11O/A
223
338
1.5
1
BuCy, TLI/lM
BuCy, TLI/lM
IVS1, llO/IVSl, 110
IVS2, l/lVS2, 1
IVS1, llO/A
364
1
BuCy, TLI/lM N37/A N37/N37
450
0.8
BuCy, TLI/l M
IVS1, llO/IVSl, 110
IVSl, 11O/A
465
476
5
2
BuCy, TLI/l M
BuCv, TLI/lG
-30/N39
IVS1, 6/FS106-7
N39/A
A/A
510
567
598
706
7
5
6
6
lG, BuTCy/lG
1G. BuTCy/lG
1G. BuTCy/lG
l G , BuTCy/lG
IVS1, 6/IVS1, 110
IVS1, 6/IVS1, 1
N39/N39
IVS1, 6/IVS1, 6
A/A
A/A
N39/A
N.4
N.4
Months
Post-BMT
% Donor
85
68
20
24
29
45
51
36
25
41
3
20
3
26
43
2
3
7
10
20
2
5
3
1
2
3
100
90
100
5
4
7
6
100
73
83
Trace
0
90
100
100
0
89
84
84
81
100
94
96
96
96
97
Clinical Outcome
Alive and well
Alive and well
Alive and well
Thalassemia intermedia
Alive and well
Alive and well
TM
Alive and well
TM
Alive
Alive
Alive
Alive
and
and
and
and
well
well
well
well
Alive and well
Abbreviations: BuCy, busulfan cyclophosphamide;TLI, total lymphoid irradiation;
BuTCy, busulfan thiotepa cyclophosphamide;
lM, CAMPATH
1M; 1G. CAMPATH 1G.
If the donor is a carrier, the detection of residual host cells
is less sensitive, because the donor DNA also carries the
mutant allele. In such situations, the level of the mutant
allele in the absence of residual host cells is already 50%;
therefore, the level of detection begins only at 53% to 55%
mutant alleles, corresponding to 5% to 10% host cells.
Where other engraftment marker techniques have failed,
the new method enabled us to demonstrate an incidence of
50% (7 of 14 patients) MC in the recipients of T cell-depleted
allografts. Furthermore, the technique demonstrated that MC
in the seven patients was stable.
In malignant hematologic diseases using a variety of engraftment marker methods, an MC incidence of 10% to 20%
has been reported after unmodified BMT.'s-20It should be
emphasized that malignant cells may persist among the donor cells in a tumor dormancy-like state. This may be misleading when interpreting data, because MC in these situations is extremely unstable.
In the nonmalignant disorder TM, MC has been evaluated
recently in 72 patients by Manna et a13 using the analysis of
the variable number of tandem repeats (VNTR). In that
study, the incidence of MC decreased with time (ie, unstable
chimerism), reaching 8% at 14 months post-BMT. However,
as also noted by these investigators, the VNTR markers do
not provide quantitative information on residual host cells.
Some investigators have reported that either high-dose irradiation or additional chemotherapy may reduce the incidence
of MC2' in a variety of diseases, whereas others found the
development of MC to be independent of the conditioning
regimen
In the above-cited study
by Manna et al.'
the conditioning regimen was shown to influence the frequency of MC post-BMT.
The present study indicates that the high incidence of MC
may be related to TCD before BMT, despite increasing the
conditioning regimen by either TLI or monoclonal anti-human lymphocyte antibody.23Indeed, TCD has been reported
to increase relapse and rejection rates in malignant diseases
because of a lack of donor anti-host lymphocyte-mediated
reaction.22324
The fact that MC has remained stable to date
(longest follow up, 68 months) suggests that MC in patients
with thalassemia need not lead to rejection of the graft or
recurrence of the disease. It should be emphasized that in
our patients with stable MC, hematopoiesis is normal, even
in those patients with only 70% donor cells. In this context,
it should also be noted that the patient with approximately
5% residual donor cells displays clinical symptoms of thalassemia intermedia at more than 4 years post-BMT. Judged
by other engraftment assays, such a patient might be considered to harbor 100% host cells.
A similar observation, ie, that stable MC permits functional donor hematopoiesis, has recently been described in
a murine model, in which correction of &thalassemia was
achieved in mice transplanted with normal congenic bone
m a r r ~ w . ~The
. ~ ' mice were conditioned by sublethal total
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D-GLOBIN MUTATION IN THALESSEMIAPOST-BMT
body irradiation, and, notwithstanding the resulting mixed
red blood cell chimerism, correction of anemia was recorded.
Quantitation of donor-type early hemapoietic progenitor
cells (CFU-S) demonstrated that the correction of the anemia
arose from a minority of normal immature bone marrow
cells. The investigators concluded that successful BMT for
@thalassemia does not necessarily require total ablation of
endogenous host cells. Because the &globin gene point mutation marker is completely independent of HLA, sex, blood
type, and VNTR, it constitutes a sensitive and accurate tool
for determination of chimerism in patients with TM after
BMT, as evidenced by the high proportion of patients with
MC detected in the present study.
In conclusion, our study suggests that the &globin gene
point mutation assay is ideal for early detection of engraftment, rejection, or recurrence of Th4 after BMT. The
incidence of MC in this report was higher than might have
been expected from the studies published to date, which
leads us to surmise that posttransplant MC with respect to
nonmalignant diseases is underreported. This may be attributed to the fact that the methods currently in use to detect
residual host cells and MC are of limited sensitivity. One
should also consider that TCD may play a critical role in
the occurrence of MC. In light of this possibility, the mode
of GVHD prophylaxis as practiced today should be reconsidered. Future studies will be required to address the issue of
cell-mediated immunotherapy to eliminate residual host cells
in patients with TM after BMT. Finally, a crucial point of
this report concerns the stable MC exhibited by our patients,
as detected by the &globin mutation assay. These findings
imply that total eradication of the host hematopoietic system
to ensure sustained engraftment in patients with TM is not
an absolute necessity.
ACKNOWLEDGMENT
We thank Merav Siani for assistance in some of the experiments
and Dr T. Pugatsch for the Y chromosome analyses.
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KAPELUSHNIK ET AL
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1995 86: 3241-3246
Analysis of beta-globin mutations shows stable mixed chimerism in
patients with thalassemia after bone marrow transplantation
J Kapelushnik, R Or, D Filon, A Nagler, G Cividalli, M Aker, E Naparstek, S Slavin and A
Oppenheim
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