Bone Marrow Transplantation, (1997) 20, 235–241
 1997 Stockton Press All rights reserved 0268–3369/97 $12.00
Persistent donor chimaerism is consistent with disease-free survival
following BMT for chronic myeloid leukaemia
N Gardiner1 , M Lawler1 , J O’Riordan1, M DeArce2, P Humphries2 and SR McCann1
1
Department of Haematology/Oncology, St James Hospital; and 2Department of Genetics, Trinity College Dublin, Ireland
Summary:
Chronic myeloid leukaemia (CML) can be treated successfully with allogeneic bone marrow transplantation
(BMT) leading to long-term disease-free survival. Leukemia relapse, however, remains a significant clinical
problem. Relapse following BMT presumably results
from the expansion of small numbers of recipient leukaemic cells which have survived the conditioning therapy. In order to define patients who are at a high risk
of leukaemia relapse, a variety of techniques have been
employed to detect persistence of host haemopoiesis
(mixed chimaerism, MC) or residual leukaemia
(minimal residual disease, MRD). However, the precise
relationship between the detection of MC and MRD
post-BMT is unknown. We have investigated chimaerism and MRD status in 22 patients who were in clinical
and haematological remission post-allogeneic BMT for
chronic phase CML. Chimaerism was assessed using
short tandem repeat PCR (STR-PCR) while BCR-ABL
mRNA detection using reverse transcriptase polymerase
chain reaction (RT-PCR) was performed to detect the
presence of MRD. Seventeen patients received unmanipulated marrow (non-TCD) while in five patients a T
cell-depleted transplant (TCD) was performed as
additional GVHD prophylaxis. Chimaerism was evaluated in 18 patients (14 non-TCD, four TCD). Mixed
chimaerism was an uncommon finding in recipients of
unmanipulated BMT (21%) when compared to TCD
BMT (100%). No evidence of MRD, as identified using
the BCR-ABL mRNA RT-PCR assay, was detected in
those patients who were donor chimaeras. Early and
transient MC and MRD was detected in four patients
(two non-TCD, two TCD) who have subsequently converted to a donor profile. One patient has stable lowlevel MC but remains MRD negative 4 years post-BMT.
Late MC and MRD was observed in two patients who
relapsed .6 years after TCD BMT for CML. We conclude that mixed chimaerism is a rare event in recipients of unmanipulated BMT and that donor chimaerism
as detected by STR-PCR assay is consistant with disease-free survival and identifies patients with a low risk
of leukaemic relapse post-BMT for CML.
Keywords: CML; BMT; chimaerism; MRD
Correspondence: Dr M Lawler, Department of Haematology/Oncology, St
James Hospital, Dublin 8, Ireland
Received 5 February 1997; accepted 17 April 1997
Allogeneic bone marrow transplantation (BMT) is a successful treatment modality for patients with chronic
myeloid leukaemia (CML). Sixty to 80 percent of patients
transplanted with unmanipulated BM in first chronic phase
(CP) achieve long-term disease-free survival.1 However,
relapse rates of 10–20% are still the most frequent cause
of treatment failure.2,3 Leukaemia relapse following BMT
presumably results from the inability of the conditioning
regimen to eliminate all recipient leukaemic cells. Ideally,
following BMT, the recipient’s bone marrow should be
completely ablated by the conditioning regimen, facilitating
the engraftment of donor haemopoietic stem cells giving
rise to a complete donor haemopoietic chimaera.4
The true incidence and significance of the detection of
mixed haemopoietic chimaerism (MC) post-BMT remains
unclear.4,5 MC was initially thought to indicate an impending relapse, however, using more sensitive molecular techniques it has become clear that MC post-BMT is not
uncommon, with varying percentages of recipient cells
being detected in different study groups.6–10 This variation
in the degree of MC is influenced by a number of factors
including the sensitivity and timing of the assay, the disease
indication for BMT, the stage of disease at time of BMT
and the choice of conditioning regimen. Several studies
indicated that low levels of persisting recipient cells are not
associated with an increased risk of leukaemic relapse.7–10
However, increasing levels of recipient cells over time
(progressive mixed chimaerism) appear to predict relapse,
especially in recipients of a T cell-depleted (TCD) transplant for CML.8,10–14
At present, PCR amplification of a DNA polymorphism
is one of the most sensitive methods for the detection of a
minor clone of recipient cells post-BMT.5 Microsatellite or
short tandem repeat (STR) regions are widespread throughout the human genome. These di-, tri- or tetra-nucleotide
repeat sequences can vary in length between individuals
and amplification of the polymorphic STRs (STR-PCR) has
been successfully used to monitor engraftment and chimaeric status of patients following allogeneic BMT.10
The persistence of leukaemic cells post-BMT is known
as minimal resiudal disease (MRD). Patients with CML
exhibit a non-random chromosomal translocation called the
Philadelphia chromosome (Ph). At the molecular level the
translocation gives rise to a novel leukaemia-specific
mRNA transcript (BCR-ABL). Reverse transcriptase polymerase chain reaction (RT-PCR) analysis of this leukaemiaspecific transcript can be used as a marker of MRD following BMT for CML. 15–22 Low levels of leukaemia-specific
Chimaerism after BMT for CML
N Gardiner et al
236
BCR-ABL mRNA transcripts are frequently detected postBMT, however, the prognostic significance of a positive
BCR-ABL PCR result is still controversial.19–23 On an individual patient basis a single positive RT-PCR result is not
indicative of relapse, however, rising levels of BCR-ABL
transcripts or continued PCR positivity is associated with
a higher relapse risk.24,25
Although chimaerism and MRD studies have indicated
that MC is associated with relapse in CML patients receiving TCD BMT, the incidence and significance of MC and
MRD status in long-term disease-free survivors of unmanipulated BMT for this disease is not clearly defined. In the
present study, patients were chosen who were in clinical
and cytogenetic remission 0.5–10 years post-allogeneic
BMT. Serial analysis was performed using STR-PCR,
nested RT-PCR for BCR-ABL mRNA and cytogenetic
analysis for the Ph chromosome to investigate the following
issues: (1) Is there a lower incidence of MC in chronic
phase CML patients receiving unmanipulated marrow as
compared to TCD marrow? (2) What is the relationship
between chimaeric status, MRD status and relapse risk following allogeneic BMT for CML? (3) Is persistent donor
chimaerism as measured by the STR-PCR assay a useful
indicator of disease-free survival (DFS) in this patient
group?
Table 1
Patients and methods
temperature from 1 to 10 years using a standard
SDS/proteinase K digestion.26 Pretransplant recipient and
donor DNA samples were amplified to identify an informative polymorphism using a panel of STR markers. The
STR-PCR protocol was as previously described. 10,26
Briefly, 20–50 ng of DNA was added to a reaction mix
containing 10 mM Tris-HCl pH 8.9, 50 mM KCl, 1% Triton
×100, 1.5 mM MgCl2, 25 pM of each primer, 200 mmol of
dATP, dGTP, dTTP and 2 mmol dCTP. Samples were
given a ‘hot start’ of 5 min at 94°C, before adding 1 U of
Taq polymerase (Perkin Elmer, Warrington, UK) and 1 mCi
a32P-dCTP and 4000 Ci/mmol (Amersham, Little Chalfont,
UK). PCR amplification had the following cycling parameters: 94°C, 1 min; 55°C, 1 min; 72°C, 1 min for 30
cycles, with a final extension step of 72°C for 3 min. To
identify the possibility of operator contamination, operator
DNA was included in each experiment. Following amplification, samples were electrophoresed on an 8% denaturing
polyacrylamide sequencing gel and results were analysed
after exposing gels to autoradiography for 4, 12 and 24 h.
The STR-PCR assay allows the detection of a minor cell
population at the 0.1–0.01% level.26 Dilution experiments
were performed to ascertain the levels of recipient cells in
MC samples. The occurrence of MC and the percentage of
recipient cells was confirmed using a second informative
STR marker.
Patients
Twenty-two patients who were transplanted for chronic
phase CML were entered into the study. All patients
received an HLA-identical sibling donor transplant in St
James’s Hospital from 1984 to 1993 and were in complete
clinical remission and at least 6 months post-BMT at the
beginning of this study. Clinical characteristics of the
patient group are outlined in Table 1. Conditioning consisted of cyclophosphamide (60 mg/kg on 2 consecutive
days) in combination with either single fraction (7.5 Gy)
total body irradiation (TBI) (n = 13), or busulphan (4 mg/kg
for 4 days) (n = 9). As graft-versus-host disease (GVHD)
prophylaxis, patients received cyclosporine (CYA) alone
(n = 8) or cyclosporine in combination with methotrexate
(CYA and MTX) (n = 14). As additional GVHD prophylaxis five patients received a T cell-depleted bone marrow
infusion. Donor marrow was obtained with informed consent on the day of transplant. Bone marrow aspirates were
taken at 3, 6 and 12 months post-BMT and thereafter yearly
for morphological cytogenetic and molecular analysis.
Two patients (UPN011/86 and UPN006/88) received
donor leukocyte infusions (DLI) for relapsed CML, 6 and
6.5 years post-T cell-depleted BMT. UPN 011/86 received
a total dose of 2.8 × 108 donor T cells/kg and UPN 006/88
received 2.9 × 108 donor T cells/kg. 5 × 106 U/m 2 interferon
a was given until days 50 and 64 post-first infusion,
respectively.
Short tandem repeat PCR
DNA was extracted from fresh bone marrow mononuclear
cells and from BM smears that had been stored at room
UPN
003/93
013/92
011/92
010/92
007/92
006/92
010/91
005/91
003/91
002/91
014/90
011/90
010/90
016/89
006/88
005/88
003/88
011/86
007/86
005/86
011/85
006/84
Clinical characteristics of patients pretransplant
Age at
Gender
Conditioning
BMT donor/recipient regimen
33
34
36
53
29
39
46
37
33
32
43
47
42
27
29
44
25
42
30
32
14
32
M/F
M/M
F/F
M/M
F/M
F/M
M/M
M/M
M/F
M/F
M/F
M/F
M/F
M/F
F/M
M/F
F/F
F/M
F/F
F/M
M/M
M/M
Bu/CY
Bu/CY
Bu/CY
TBI/CY
TBI/CY
TBI/CY
TBI/CY
TBI/CY
TBI/CY
TBI/CY
Bu/CY
Bu/CY
Bu/CY
TBI/CY
TBI/CY
TBI/CY
TBI/CY
TBI/CY
TBI/CY
TBI/CY
Bu/CY
TBI/CY
GVHD
prophylaxis
T cell
depletion
CYA + MTX
CYA + MTX
CYA + MTX
CYA + MTX
CYA + MTX
CYA + MTX
CYA + MTX
CYA + MTX
CYA + MTX
CYA + MTX
CYA + MTX
CYA + MTX
CYA + MTX
CYA + MTX
CYA
CYA
CYA
CYA
CYA
CYA
CYA
CYA
No
No
No
No
No
No
No
No
No
No
No
No
No
No
Yes
Yes
Yes
Yes
Yes
No
No
No
CY = cyclophosphamide; Bu = busulphan; CYA = cyclosporine; MTX
= methotrexate.
Cytogenetic analysis
Bone marrow aspirates were performed at varying intervals
post-BMT. Karyotyping was performed using standard
techniques. A minimum of 25 metaphases was analysed per
sample. This allows the detection of a minor clone at the
5% level.
Chimaerism after BMT for CML
N Gardiner et al
RT-PCR analysis
RNA was extracted from frozen BM and PB samples following red cell lysis using the Chomczynski method.27
Where cell pellets were less than 1 × 106 cells, 2 mg of glycogen (Boehringer Mannheim, Lewes, UK) was added as
an RNA carrier. Two micrograms of RNA were treated
with DNAse I (Gibco BRL, Paisley, UK) and reverse transcribed using MMLV reverse transcriptase (Gibco BRL,
Paisley, UK) following the manufacturer’s recommendations. Strict contamination avoidance procedures were
followed and control reactions were added at each stage
of the RNA procedure. Two-step nested PCR analysis was
performed using the protocol described by Roth et al.16,19
The amplification protocol was adapted to accommodate
the use of dUTP in place of dTTP (PCR Carryover Prevention Kit; Perkin Elmer). The incorporation of uracil into the
PCR reaction products (amplicons) enables all subsequent
primary and nested PCR mixes to be treated with uracilN-glycosylase (UNG) which will cleave any contaminating
amplicons.28 The UNG is subsequently denatured at 94°C
for 3 min, before adding cDNA. The use of the PCR carryover prevention kit successfully eliminated any further false
positive results.
The PCR products were visualised on ethidium bromidestained 2% agarose gels. The product of the b3a2 transcript
is 300 bp in size and the b2a2 transcript gives rise to a
smaller 225 bp product. RNA integrity was assessed in all
samples by amplification of the ABL gene. The routine sensitivity of the RT-PCR was 1:105. Samples were only
scored as positive or negative if the dilute positive control
(1 K562 cell: 105 HL60 cells) amplified after nested PCR
and all contamination controls were negative.
Results
Chimaerism results
Eighteen of the 22 patients were studied from 6 months to
10 years post-BMT. Chimaeric assessment was not possible
in four patients as no pretransplant and/or donor material
was available for analysis. A median of five analyses (range
2–6) was performed for each patient post-BMT (Figure 1
and Table 2).
Donor chimaerism: Eleven of 18 patients assessed (61%)
were exclusively donor chimaeras at all times post-BMT
(UPNs 011/85, 005/86, 016/89, 011/90, 014/90, 002/91,
010/91, 007/92, 010/92, 011/92, 013/92). (see Figure 1 and
Table 2). These patients all received an unmanipulated
BMT, thus donor chimaerism was observed in 78% (11/14)
of patients receiving an unmanipulated BMT while 0/4
recipients of T cell-depleted transplants exhibited a complete donor profile at all times tested (Table 2).
Mixed chimaerism: Seven of 18 patients studied exhibited
MC post-BMT (07/86, 011/86, 05/88, 06/88, 03/91, 05/91,
06/92) (see Figure 1 and Table 2). Within the group of 14
patients who received an unmanipulated BMT, only three
patients were mixed chimaeras (UPNs 003/91, 005/91
006/92). UPN 003/91 had recipient cells present at 3
months post-BMT but was fully donor at 6 months and
on all subsequent analyses. UPN 005/91 showed low-level
mixed chimaerism at all times post-BMT. At 1 year postBMT, 10% recipient cells were present. Low levels of ,5%
recipient cells persist to the last follow-up sample at 4 years
post-BMT. UPN 006/92 showed evidence of recipient cells
up to 2 years post-BMT. The levels of recipient cells
decreased with time and analysis at 3 years post-BMT
showed an exclusively donor profile.
Four of four patients studied who received a T celldepleted BMT exhibited MC (007/86, 011/86, 005/88,
006/88). UPN 007/86 had evidence of recipient cells within
the first year post-BMT but exhibited a donor profile on
subsequent analyses. UPN 005/88 showed a mixed chimaeric profile at 1 and 3 years post-BMT. Analysis from 4 to
7 years post-BMT demonstrated a donor profile. UPN
011/86 was a mixed chimaera with 5% recipient cells
detected at 5 years post-BMT, 1 year prior to haematologic
relapse. Subsequent chimaerism analysis at the time of
relapse indicated the presence of .95% recipient cells.
UPN 006/88 had not been assessed for chimaeric status
prior to relapse but .95% recipient cells were detected at
time of full haematological relapse. Donor leukocyte
infusions resulted in the elimination of the recipient cells
and the establishment of a complete donor chimaeric profile
by day 91 post first infusion in both patients.
BCR-ABL mRNA assessment results
Twenty of 22 patients were assessed for MRD using RTPCR for BCR-ABL mRNA. Follow up ranged from 6
months to 10 years post-BMT and patients were assessed
a median of three times (range 1–6) (Figure 1).
BCR-ABL mRNA negative: Fourteen of 20 patients showed
no evidence of BCR-ABL mRNA at any time post-BMT
with a follow-up of .5 years in 10 patients (Figure 1). Seventeen of 20 patients (85%) had no detectable BCR-ABL
positive clone at a sensitivity level of 10−5, 1 year postBMT and remained PCR negative on all subsequent analyses. This group included 13/15 patients who received an
unmanipulated BMT.
BCR-ABL mRNA positive: Early BCR-ABL positivity was
seen in two patients (UPN 002/91 and 003/91) 6 weeks and
3 months post-BMT. In each patient, follow-up RT-PCR
analysis was negative at 2 and 4 years post-BMT, respectively. One patient (UPN 005/91) had a positive PCR result
3 years post-BMT, the follow-up samples at 4–7 years were
RT-PCR negative. Two T cell-depleted BMT recipients
relapsed; UPN 011/86 had a PCR positive result 10 months
prior to haematological relapse. UPN 006/88 was negative
2 years before haematological relapse, but no subsequent
sample was available for analysis. Analysis at 6 months
post-DLI therapy indicated that the leukaemic clone had
been eliminated.
Cytogenetic results
Cytogenetic analyses were performed on all 22 patients
with a median of five analyses (range 2–9). Philadelphia
237
Chimaerism after BMT for CML
N Gardiner et al
238
003/93
*
*
013/92
INDEX
011/92
Philadelphia positive
Philadelphia negative
010/92
*
*
*
BCR/ABL positive
*
007/92
BCR/ABL negative
*
006/92
Donor chimera
Mixed chimera <10% recipient cells
010/91
C
Mixed chimera >20% recipient cells
005/91
* Donor sex marker
003/91
002/91
*
*
*
*
*
*
*
*
C Cytogenetic relapse
H Haematological relapse
*
DLI Donor leukocyte infusions
# T cell-depleted BMT recipients
014/90
*
011/90
*
*
*
*
010/90
*
016/89
*
*
*
*
*
H
#006/88
*
#005/88
*
*
*
*
*
DLI
*
C
*
*
*
*
*
*
#003/88
*
#011/86
*
*
*
*
H
DLI
*
*
*
#007/86
005/86
*
011/85
006/84
1
2
3
4
5
6
7
8
9
10
Years post-BMT
Figure 1
Analysis of chimaeric status and MRD in CML patients post-allogeneic BMT.
(Ph)-positive metaphases were not detected in 18/22
patients (82%) at any time post-BMT (Figure 1). UPN
005/91 had 30% Ph-positive metaphases at 1 year postBMT; this Ph-positive clone disappeared spontaneously and
the patient has been Ph negative on all subsequent analyses.
UPN 005/88 who received a T cell-depleted BMT, showed
a transient appearance of 3% Ph metaphases at 3 years postBMT but remains Ph negative 7 years post-BMT. In UPN
011/86 and 006/88 the first evidence of Ph-positive metaphases was at the time of haematological relapse 6 and 6.5
years post-BMT. A cytogenetic remission was seen at day
84 (UPN 011/86) and day 98 (UPN 006/88) post first donor
leucocyte infusion.
MC, MRD and DFS
Twenty of 22 patients remain in clinical remission 3–10
years post-BMT with a median follow-up of 5.5 years.
Eleven of 18 patients available for analysis by STR-PCR
who were donor chimaeras all remain in remission and
show no evidence of MRD (Table 2). Four patients who
were initially mixed chimaeras but converted to full donor
chimaerism up to 4 years post-BMT, also show no evidence
of recurrent disease. BCR-ABL positivity was found in one
of these patients (UPN 005/88) 3 years post-T cell-depleted
BMT, coinciding with a transient cytogenetic relapse (3%
Ph-positive metaphases). Subsequent analysis indicated the
Chimaerism after BMT for CML
N Gardiner et al
Table 2
Patient characteristics post-transplant
UPN
Chimaerism
<1 year
003/93
013/92
011/92
010/92
007/92
006/92
010/91
005/91
003/91
002/91
014/90
011/90
010/90
016/89
006/88a
005/88a
003/88a
011/86a
007/86a
005/86
011/85
006/84
—
DC
DC
DC
DC
MC
DC
MC
MC
DC
DC
DC
—
DC
—
MC
—
—
MC
—
—
—
MRD
GVHD
Status
Grade II
None
Grade III
Grade I
None
None
Grade II
Grade II
Grade I
None
Grade II
Grade I
None
None
Grade I
None
None
Grade III
Grade I
Grade III
Grade I
Grade II
DF
DF
DF
DF
DF
DF
DF
CyR→DF
DF
DF
DF
DF
DF
DF
HR
CyR→DF
DF
HR
DF
DF
DF
DF
.1 year
—
neg
DC
neg
DC
neg
DC
—
DC
neg
MC→DC
neg
DC
—
MC
neg
DC
Pos→neg
DC
Pos→neg
DC
neg
DC
neg
—
neg
DC
neg
MC
pos
MC→DC pos→neg
—
neg
MC
pos
DC
neg
DC
neg
DC
neg
—
neg
a
Patients who received T cell-depleted BMT.
DC = donor chimaerism; MC = mixed chimaerism; HR = haematological
relapse; CyR = cytogenetic relapse; DF = disease free.
spontaneous disappearance of the leukaemic clone and the
re-establishment of donor haemopoiesis.
The three remaining patients (UPNs 011/86, 006/88,
005/91) showed persistent and/or late MC. UPN 005/91 had
evidence of a cytogenetic relapse at 1 year post-BMT. STRPCR indicated approximately 10% recipient cells at this
time. The leukaemic clone (as assessed by chromosomal
and RT-PCR analysis) subsequently disappeared without
additional treatment, but low-level MC persists in this
patient up to 4 years post-BMT. The patient remains in
clinical and cytogenetic remission.
UPN 011/86 relapsed 6 years post-T cell-depleted BMT.
Retrospective analysis indicated that 5% recipient cells and
BCR-ABL positivity was present 1 year prior to relapse.
UPN 006/88 relapsed 6.5 years post-T cell-depleted BMT.
No MRD was detected 2 years prior to relapse and material
was not available for analysis 1 year prior to relapse. STRPCR indicated a predominantly recipient haemopoiesis
(95–99% recipient cells/1–5% donor cells) at time of
haematological relapse in both patients. Both patients
responded to DLI therapy and remain in full haematological
and molecular remission.
Discussion
Chimaeric studies in patients undergoing BMT for CML
have indicated that MC is a common event but the majority
of studies have involved T cell-depleted BMT recipients. 5
None the less, these results have indicated a putative association between MC, the presence of MRD and risk of leukaemia relapse.7–9,14 Roux et al29 found that 7/8 (87%) of
CML patients who were mixed chimaeras following T celldepleted BMT relapsed, compared to 0/8 (0%) of donor
chimaeras.
In the present study MC was a rare event in long-term
survivors following unmanipulated BMT for chronic phase
CML. Eleven of 14 patients (79%) assessed at serial timepoints post-unmanipulated BMT for chronic phase CML
were donor chimaeras. Three of 14 patients (21%) were
mixed chimaeras. Two of these patients reverted to donor
chimaerism and one patient had persistent MC at 4 years
post-BMT. Thus 13/14 recipients (93%) of unmanipulated
BMT exhibited donor chimaerism at the end of the study
period. These results contrast strongly with the incidence
of mixed chimaerism in the T cell-depleted BMT group.
Four of 4 patients (100%) who received a T cell-depleted
BMT exhibited MC post-BMT. Two patients converted to
donor chimaerism while two patients had late haematological relapses with predominantly recipient haemopoiesis.
Elmaagacli et al30 reported a surprisingly high incidence
of MC (64% within 24 months of BMT and 53% after 24
months post-BMT) in 28 male recipients of female marrow
post-unmanipulated BMT for CML. However, when
patients transplanted in blast crisis or accelerated phase and
those who received in vivo T cell depletion for a matched
unrelated BMT are removed from the analysis, the incidence of MC, 3/12 patients (25%), is similar to our own
study. Thus the incidence of MC following unmanipulated
BMT for CP-CML is approximately 20% and this is substantially lower than the 50–100% incidence report by different
studies following T cell-depleted BMT for CML.8–10,13,14
Examination of the incidence of MRD in recipients of
unmanipulated vs T cell-depleted marrow shows a similar
pattern; two of 15 recipients (13%) of unmanipulated BMT
exhibiting transient MRD positivity as compared to 3/5
(60%) MRD positivity in the T cell-depleted group. This
lower incidence of BCR-ABL mRNA positivity in the
recipients of non-T cell-depleted BMT is in agreement with
other MRD studies.22
The high incidence of donor chimaerism and/or MRD
negativity at 1 year post-BMT (86% in non-TCD BMT
recipients, 77% in the overall group) is reflected in the DFS
of this patient cohort with no relapses observed in donor
chimaeras/MRD negative patients, 3–10 years post-BMT.
Similarly, the transient appearance of a minor subpopulation of recipient Ph-positive BCR-ABL expressing cells
in UPN 005/91 (at 1 year) and UPN 005/88 (at 3 years)
was not associated with frank haematological relapse. Lin
et al31 recently reported transient cytogenetic relapse
involving the eradication of the Ph clone without clinical
intervention in 4/98 patients following BMT for CML.
However, of these four patients, two subsequently progressed to haematological relapse while two remain BCRABL positive and presumably at risk of relapse. UPN
005/91 and UPN 5/88 remain Ph and BCR-ABL negative 3
and 4 years post-transient cytogenetic relapse, respectively,
without clinical intervention. Two patients (UPNs 0011/86
and 006/88) relapsed 6.5 and 6 years following TCD BMT
for CML. Both patients had predominantly recipient
haemopoiesis and were BCR-ABL positive at time of
relapse. They responded to donor lymphocyte infusions
(DLI) and remain in full haematological and molecular
239
Chimaerism after BMT for CML
N Gardiner et al
240
remission as judged by cytogenetics, STR-PCR and RTPCR for BCR-ABL. Thus, donor chimaerism is associated
with MRD negativity and DFS. Transient MC/MRD positivity and low-level MC is associated with a low risk of
haematological relapse particularly following unmanipulated BMT for CP CML.
RT-PCR for BCR-ABL mRNA transcript, with a routine
sensitivity of 10−5 , is presently the most sensitive MRD
detection assay system. However, RT-PCR is subject to
varying efficiencies particularly with hypocellular samples.
In addition, two-step PCR protocols makes the procedure
extremely prone to contamination and require extensive
precautionary procedures. In comparison, the STR-PCR
assay is a relatively simple and robust molecular technique
which can be used for monitoring all allogeneic BMT
recipients and is especially useful in sex-matched BMT.
Using a radioactive label (dCTP32a) the STR-PCR assay
has a routine detection sensitivity of 0.1–0.01%. The
quantification of recipient cell levels by measuring the relative intensities of the STR bands and analysis of consecutive samples in a given patient, allows the relative decrease
or increase of recipient cell levels to be determined without
recourse to technically difficult quantitative PCR assays.
While we agree that nested RT-PCR for the detection of
BCR-ABL is the most sensitive method to detect MRD,
we suggest that STR-PCR may be used as an appropriate
screening test; if residual recipient cells are detected using
this methodology, patients should be monitored more rigorously using quantitative RT-PCR.
In conclusion, serial STR-PCR has indicated: (1) mixed
haemopoietic chimaerism post-unmanipulated BMT for CP
CML is a rare event; and (2) donor chimaerism is consistent
with the elimination of MRD and DFS.
Acknowledgements
This work was supported by the Cancer Research Advancement
Board and the Health Research Board of Ireland.
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