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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 95 No. 8 (April 15), 2000:
pp. 2659-2665
NEOPLASIA
Molecular analysis of lineage-specific chimerism and minimal
residual disease by RT-PCR of p210BCR-ABL and
p190BCR-ABL after allogeneic bone marrow transplantation
for chronic myeloid leukemia: increasing mixed myeloid chimerism and
p190BCR-ABL detection precede cytogenetic relapse
Josefina Serrano,
Jose Roman,
Joaquin Sanchez,
Antonio Jimenez,
Juan A. Castillejo,
Concepcion Herrera,
Maria Gracia Gonzalez,
Luisa Reina,
Maria del Carmen Rodriguez,
Miguel A. Alvarez,
Juan Maldonado, and
Antonio Torres
From the Hematology Department of Reina Sofía Hospital,
Córdoba, Spain, and the Hematology Department of Carlos Haya
Hospital, Malaga, Spain.
 |
Abstract |
We studied lineage-specific chimerism and minimal residual disease
(MRD) in sequential posttransplant samples from 55 patients who
underwent unmanipulated (n = 44) or partially T-cell-depleted (n = 11) allogeneic bone marrow transplantation (BMT) for chronic myeloid leukemia (CML). Chimerism was assessed by
polymerase chain reaction (VNTR [variable number of
tandem repeats]-PCR) analysis in highly purified CD19+, CD3+,
CD15+, and CD56+ cell fractions, whereas MRD was investigated in
whole blood by reverse transcriptase-PCR (RT-PCR) of both
p210BCR-ABL and p190BCR-ABL hybrid transcripts.
Of 55 patients, 14 (including 6 T-cell-depleted patients) had
cytogenetic relapse at 5-80 months and progressed to hematologic
relapse, while 41 patients remained in prolonged cytogenetic
remission 12-107 months post-BMT. Before leukemia recurrence, patients
in the relapse group showed a consistent evolution pattern sequentially
featured by persistent p210BCR-ABL positivity, increasing
mixed chimerism (MC) in myeloid cells, p190BCR-ABL positivity, and, finally, cytogenetic
relapse. Myeloid MC preceded cytogenetic relapse by 2-12 months,
whereas p190BCR/ABL was detected 1-6 months prior to
cytogenetic relapse in 11 patients and concomitant with cytogenetic
relapse in 3 patients. In the remission group, all patients invariably
tested negative for p190BCR-ABL; 10 patients tested
positive for p210BCR-ABL at variable time-points but showed
persistent full donor chimerism (DC), whereas 31 patients tested
p210BCR-ABL negative and displayed full DC or transient MC
due to the persistence of recipient T cells. Two patients in the
relapse group were successfully reinduced into molecular remission with
donor lymphocyte infusion. Sequential molecular analysis after such
treatment showed the inverse pattern to that observed prior to relapse,
ie, progressive disappearance of p190BCR-ABL transcripts,
conversion of myeloid chimerism to donor type, and, finally,
p210BCR-ABL negativity. We conclude that lineage-specific
chimerism and p190BCR-ABL messenger RNA (mRNA) analyses
contribute a better characterization of CML evolution after BMT and
enable early identification of patients at the highest risk of relapse.
(Blood. 2000;95:2659-2665)
© 2000 by The American Society of Hematology.
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Introduction |
Although allogeneic bone marrow transplantation (BMT)
may cure 50% to 60% of patients with chronic myeloid leukemia (CML), disease relapse still represents the major cause of treatment failure.1,2 Patients who relapse after BMT may be reinduced into durable second remission with distinct therapeutic strategies including  -2a-interferon (INF) and/or donor lymphocyte infusion (DLI).3-6 Among prognostic determinants of response to
salvage therapy, disease burden appears to represent a significant
factor.7,8 Thus, minimal residual disease (MRD)
evaluation aimed at early detection of relapse has relevant therapeutic
implications in this context.
Monitoring of MRD after BMT for CML has mainly involved a karyotypic
search for the Philadelphia (Ph1) chromosome and/or reverse transcriptase-polymerase chain reaction (RT-PCR) amplification of the
p210BCR-ABL hybrid transcript. Detection of the Ph1
chromosome or p210BCR-ABL messenger RNA (mRNA) after 6 or
more months post-BMT has been associated with subsequent hematologic
relapse.9-11 Conventional karyotyping has limited
sensitivity, whereas RT-PCR enables smaller amounts of MRD to be
detected and might permit therapeutic intervention at an earlier stage.
However, after BMT a sizable proportion of patients in long-term
remission test p210BCR-ABL positive,12,13 and
the issue of whether or not molecular relapse should be treated remains
unresolved. Recently quantitative PCR analysis of
p210BCR-ABL has provided more precise prognostic
information on post-BMT disease evolution in CML patients, and it is
likely that this technology will aid therapeutic decision making in the
future.14,15
As a further approach to monitoring post-BMT outcome, several
investigators have employed chimerism analysis using
chromosome Y body or highly polymorphic loci detection.
These techniques permit the relative proportions of host and donor
cells in the post-BMT period to be identified and quantified. Although
chimerism analysis cannot directly detect residual leukemia, it
permits the definition of specific patterns, such as increasing
autologous hemopoiesis (mixed chimerism [MC]), that are known to be
associated with disease recurrence.16-18 On the other hand,
MC detection in whole blood or marrow may be transient if it is the
result of residual host T cells that have survived the conditioning
regimen. This drawback could be overcome by analyzing lineage-specific chimerism in highly purified cell fractions (myeloid, B and T lymphocytes, and natural killer [NK] cells).
Recently the p190BCR-ABL transcript, which is classically
associated with Ph1-positive acute lymphocyte leukemia (ALL), has been detected at diagnosis in virtually all patients with CML, where it
occurs as a consequence of alternative or missplicing events in the BCR
gene.19,20 Because the amount of p190BCR-ABL
has been correlated with that of p210BCR-ABL, corresponding
to 0.02%-30% of the total BCR-ABL transcripts, one could speculate
that a rising tumor burden is needed for p190BCR-ABL to
become detected by RT-PCR during hematologic remission. Therefore we
tested the hypothesis that p190BCR-ABL mRNA detection could
be used, in addition to other markers, as an indicator of disease
evolution in the post-BMT outcome of CML patients.
We report here a molecular study that was performed on a series
of CML patients who underwent a BMT. The study used
p210BCR-ABL and p190BCR-ABL RT-PCR combined
to lineage-specific chimerism analysis in highly purified cell
fractions. We found that this strategy closely traces the kinetics of
leukemic regrowth after allogeneic BMT. In fact, disease evolution in
relapsed patients was consistently characterized by the sequential
detection of p210BCR-ABL transcripts; increasing amounts of
myeloid MC; and finally, p190BCR-ABL positivity preceding
impending cytogenetic and hematologic recurrence. This model was
further reinforced by an inverse sequence of molecular findings
detected after DLI treatment for relapse in 2 patients, both of whom
experienced progressive disappearance of p190BCR-ABL,
chimerism pattern conversion to donor type, and p210BCR-ABL negativity.
| |
Materials and methods |
Patients
From June 1989 to December 1997, 55 consecutive recipients with
Ph1-positive CML presented with both p210BCR-ABL and
p190BCR-ABL mRNA types at diagnosis and just before
transplantation. All patients underwent allogeneic BMT from human
leukocyte antigen-identical (HLA-identical) siblings (n = 50) or
HLA-matched unrelated volunteers (n = 5) at the Reina Sofia Hospital,
Córdoba, Spain, and the Carlos Haya Hospital, Malaga, Spain.
Partially T-cell-depleted marrow grafts containing
1 × 106 donor T-CD8+ cells/kg were implanted in 11 patients as previously described,21 whereas 44 patients
received unmanipulated BMT. The main patient characteristics at
the time of BMT, including disease phase, conditioning regimen, and
graft-versus-host disease (GVHD) prophylaxis, are reported in Table
1. Results were analyzed from December 31, 1998.
Sample collection and manipulation
Conventional karyotyping, chimerism analyses, and
p210BCR-ABL RT-PCR studies were prospectively performed in
diagnostic material and in sequential post-BMT blood or marrow samples
collected at monthly intervals during the first year, at 3-month
intervals during the second year, and at 6-month intervals thereafter.
Viable cells were also cryopreserved at each time point.
Lineage-specific chimerism and p190BCR-ABL RT-PCR were
analyzed in cryopreserved samples collected before September 1996 and
prospectively in fresh samples thereafter.
Cell separation of the myeloid and lymphoid subsets was performed using
immunomagnetic beads (Dynal, Oslo, Norway) either coupled with the
specific antibody (CD15, CD3, and CD19) or coupled with an
antimouse immunoglobulin G (IgG) for purification of the NK fraction
previously incubated with an anti-CD56 antibody (Becton Dickinson, San
Jose, CA). Separation procedures were performed according to the
manufacturer's instructions. Purity was assessed using a direct
immunofluorescence technique with specific monoclonal antibodies (mAbs)
conjugated with fluorescein isothiocyanate (FITC) or R-phycoerythrin
(R-PE) (Becton Dickinson). Acquisition and analysis were
performed in a cytometer (FACScalibur, Becton Dickinson). Cells were
only further processed when purity was at least 98%. DNA of distinct
cell populations was isolated using a modified salting-out
procedure.22 VNTR analysis was performed as described below.
Minimal residual disease of p210BCR-ABL and
p190BCR-ABL detected by RT-PCR
Total RNA was isolated from mononuclear peripheral blood or bone
marrow cells as described by Chomczynski and Sacchi.23 Reverse transcription was performed by first heating 1 µg total RNA
at 70°C for 5 minutes then using the heated RNA with random hexamers as reaction primers. The reaction was carried out at 42°C
for 45 minutes in the presence of 12 units of avian myeloblastosis virus (AMV) reverse transcriptase. The method used to amplify in 2 different "nested" PCRs, respectively, the b2a2/b3a2 types of
BCR-ABL hybrid mRNA and the e1a2 type, was performed essentially as
described by Saglio et al.20 The second-round PCR products were electrophoresed on a 1.5% agarose gel containing 1 µg/mL ethidium bromide and photographed under ultraviolet (UV) light. Precautions were taken to ensure high PCR quality as recommended by
Kwok and Higuchi.24 Using this approach, we could detect a
single BCR-ABL positive cell in 105 normal
cells.11
Patients who always had positive BCR-ABL results or who had both
BCR-ABL positive and negative time points and a positive last sample
were considered as BCR-ABL positive patients. Recipients who presented
BCR-ABL negative assays or presented initial positive assays followed
by negative results on subsequent analyses were considered as BCR-ABL
negative patients.
Chimerism assessment by VNTR-PCR analysis
High molecular weight DNA was extracted from donor and recipient
mononuclear cells using a salting-out procedure.25 For PCR
amplification, we used specific primers designed to flank the repeat
units of the following human minisatellite regions (VNTR-PCR): D1S80,
33.6, 33.1, 33.4, YNZ-22, APO-B,  g3, DXS52, and HVR-3'. Primer
sequences and conditions for each reaction have been described
elsewhere.26 We defined a VNTR locus as being informative
if the analysis of recipient and donor samples prior to BMT showed a
unique band for the recipient and another unique band for the donor or
if it showed a unique band for the recipient only. The VNTR-PCR assay
allows the detection of a minor cell population at the 0.5% to 1.5%
level.26
Patients who exhibited complete donor hematopoiesis with at least 2 markers were defined as having full donor chimerism (DC). Patients who
exhibited mixed populations of donor and host cells with at least 2 markers on more than 1 occasion were considered as having MC (Figure
1).
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| Fig 1.
Amplification of the D1S80 locus in 2 patients (UPNs 322 and 306) electrophoresed in 1.5% agarose gel.
Lines 2 and 5 depict recipient samples before transplantation; lines 3 and 6 depict donor samples. An MC profile is observed in the total
white cell fraction at +8 months post-BMT in patient UPN 322 (line 4).
A complete donor profile is observed in the follow-up of patient UPN
306 at +20 months post-BMT (line 7). Line 1 depicts water, which is a
blank control.
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To evaluate the possible dynamics of chimerism after BMT, we
established a quantitative PCR approach. Briefly, recipient
pretransplant DNA and donor genomic DNA were mixed in different
percentages and were subsequently amplified. PCR products were stained
with ethidium bromide after separation by agarose gel electrophoresis (Figure 2A). Light emissions of the gels
were directly digitalized under UV-light stimulation and subsequently
analyzed densitometrically (Bio-1D software, Vilber Lourmat,
Marne-La-Vallee, France). The peak areas of donor and recipient bands
directly correlated to DNA concentrations. After determining the
percentage of host signal intensities, standard curves for each
individual patient were generated. Signal intensities of individual
samples were related to the patient's standard curve (Figure 2B). The
degree of MC was expressed as the percentage of host DNA. We prepared
standardized chimeric samples by mixing pretransplant recipient and
donor DNA in dilution experiments (75% to 0.39%) in relation to host
DNA. Each sample was amplified with the informative primer. The
intensity of each allele was measured densitometrically. The proportion of host DNA was expressed according to the quotient, where
peak area recipient/peak area donor equals quotient host/donor DNA.
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| Fig 2.
Generating and plotting individual standard curves.
(A) Mixing experiments for patient UPN 251 by PCR with D1S80 primers
for generating an individual standard curve. Recipient (R) DNA was
mixed with donor (D) DNA in various proportions as indicated. (B)
Evaluating locus D1S80 of patient UPN 251 for generating the standard
logarithmic curve. After amplification and densitometric analysis of
the chimeric donor and recipient samples, a standard curve was
generated by plotting the percent recipient DNA versus the mean values
of quotient recipient/donor DNA.
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The quotient host DNA was correlated to the percentage of host DNA
given to the chimeric samples. The mixing experiments were carried out
in at least duplicate, and standard curves were then generated from
mean values. The analysis of each prior posttransplant sample was
repeated when an actual blood sample was received. In general,
measurements were completed 5-12 times, and mean values were calculated
from repeated analyses.
Cytogenetic analyses
For cytogenetic analyses, cells from bone marrow were cultured in
McCoy's medium supplemented with 20% fetal calf serum and antibiotics
for 48 hours. Chromosome preparations were stained with 5% Giemsa
solution according to standard procedures. A minimum of 25 metaphases
were analyzed per sample. This allowed the detection of a minor clone
at the 5% level. Cytogenetic relapse was considered to have occurred
if one or more Ph1 positive metaphases were detected at any time
following BMT without evidence of hematological relapse.
Statistical analysis
Cumulative actuarial probabilities (plus or minus standard errors)
were calculated by the product-limit method.27 Differences between time-to-event distribution functions were compared by the
log-rank test. Comparisons between continuous covariates were performed
by the 2-tailed Wilcoxon signed rank test across strata. Differences
between frequencies were compared by the 2-tailed Fisher exact test.
Stepwise proportional hazards general linear model analysis was used to
evaluated the effect of different covariates on the analytical
endpoints of cytogenetic relapse and p190BCR-ABL
expression.28
| |
Results |
Post-BMT outcome
During the post-BMT period, 41 patients remained in cytogenetic and
hematologic remission at a median duration of 34 months (range:
12.5-107 months), and 14 patients (including 6 T-cell- depleted
patients) experienced cytogenetic relapse at a median duration of 10.5 months (range: 5-80 months). Cytogenetic relapse was followed in all
cases by hematologic relapse at a median duration of 2 months (range:
1-11 months). Of the 14 patients with cytogenetic relapse, 10 patients
were in the chronic phase, 2 in the accelerated phase, and 2 in the
blastic phase. The 2 patients with blastic phase relapse were given
chemotherapy, and the 12 patients with chronic/accelerated phase
relapse received -INF at the time of hematologic recurrence. Of
these latter 12 patients, 3 patients (unique patient numbers [UPNs]
235, 251, and 2056) were also treated with donor leukocyte infusion
(DLI) between 7-36 months post-BMT, and 1 patient (UPN 199) underwent a
second BMT.
Molecular monitoring results in patients in cytogenetic and
hematologic remission
Of the 41 patients in this group, 31 patients tested
p210BCR-ABL negative, 28 had signs or symptoms of
persistent full DC, and 3 had a transient MC pattern at low levels
(less than 30%) in whole blood, which lasted 4-5 months. Chimerism
studies on purified cell fractions in these latter cases showed
persistent recipient-type CD3+ cells and full DC in CD15+, CD19+, and
CD56+ cell fractions. The chimerism pattern of the T-cell fraction
converted to the donor type in subsequent evaluations of these 3 patients.
There were 10 patients (all of whom had received unmanipulated BMT) who
tested p210BCR-ABL positive at various time points and in
the last follow-up control. All 10 patients displayed persistent full
DC in whole blood as well as in purified cell populations. All 41 patients who remained in cytogenetic and molecular remission
tested persistently negative for the p190BCR-ABL mRNA by
RT-PCR.
Molecular monitoring results in patients undergoing cytogenetic and
hematologic relapse
The results of molecular monitoring in this group are shown in Table
2. All 14 patients persisted (n = 6) or
converted (n = 8) to a positive p210BCR-ABL at 1-62 months following BMT. Results of sequential chimerism analyses in whole
blood and purified cell fractions were as follows.
MC was detected in whole blood at 1-68 months in 13 cases; this
preceded p210BCR-ABL mRNA detection in 1 patient (UPN 322, Table 2), was observed concurrently with p210BCR-ABL in 3 cases (UPNs 245, 305, and 2072), and followed p210BCR-ABL
detection by 1-6 months in 9 cases. Chimerism analysis in purified cell
fractions revealed myeloid MC (detected in CD15+ cells) simultaneously with whole blood MC in 12 patients. In 1 patient (UPN 322), myeloid MC
was detected 6 months after MC was detected in the whole blood. Longitudinal myeloid MC quantitative evaluation showed a progressive increase of autologous DNA in these 13 patients (Table 2). Increasing myeloid MC preceded cytogenetic and hematologic relapse by 2-12 months
and 3-16 months, respectively. Chimerism analysis of the CD3+ fraction showed only donor-type T cells in 8 patients
at the first detection of MC. Interestingly, this molecular profile with full-donor origin in T-cell fraction persisted in 4 of these 8 patients at the time of cytogenetic relapse (UPNs 235, 251, 267, and 2056).
Chimerism evolution in CD19+ and CD56+ cell
fractions was not too different from that observed in the
CD3+ subset, although an earlier reappearance of recipient
cells in patients who presented T-cell MC was detected. One patient
(UPN 286) had a persistent full DC pattern in whole blood and
fractionated cell populations. This patient underwent an unusual
pattern of hematologic relapse in donor cells.
All 14 patients (including UPN 286) had a positive PCR conversion to
p190BCR-ABL at 4-80 months post-BMT. The detection of
p190BCR-ABL followed p210BCR-ABL detection in
all but 1 case (UPN 267), and in 12 of 14 cases MC was detected.
RT-PCR positivity for p190BCR-ABL preceded cytogenetic
relapse by 1-6 months in 11 patients and was detected simultaneously
with cytogenetic recurrence in 3 patients.
Molecular kinetics after treatment of relapse
No major cytogenetic and molecular changes were observed after
treatment of relapse in patients receiving INF. The complete disappearance of p210BCR-ABL and p190BCR-ABL
transcripts and chimerism conversion to donor type were documented in
one patient (UPN 199) after the second BMT. The 3 patients who received
DLI following INF were also prospectively monitored. In 1 patient (UPN
235) who received a single dose of 1 × 107 CD3
cells/kg, the positive RT-PCR for both p210BCR-ABL
and p190BCR-ABL transcripts persisted, and there was
increasing autologous hemopoiesis. This patient refused further
treatment and remains currently in the second CML chronic phase.
Complete hematologic and cytogenetic response following DLI was noted
in 2 patients. One patient (UPN 251) received 2 escalating T-cell doses
(1 × 107 and
1 × 108 CD3 cells/kg) and an infusion of
1.5 × 106 and 4.5 × 106 CD34
cells/kg in an attempt to abrogate post-DLI aplasia. A transient response was observed after the first dose, with total disappearance of
recipient T cells and a 2-fold decrease of recipient granulocytes. After the second infusion, sequential molecular analyses showed the
inverse pattern observed prior to relapse, ie, progressive negativity
of p190BCR-ABL RT-PCR (the day of infusion plus 15 days
[day +15] following the second DLI), myeloid cell
chimerism conversion to donor type (day +90), and finally disappearance
of p210BCR-ABL transcripts at day +120 (Figure
3). One patient (UPN 2056) did not
experience a hematologic response following the first DLI dose of
1 × 107 CD3 cells/kg, and a second dose of
1 × 108 CD3 cells/kg was infused. Despite the
concomitant administration of 1.3 × 106 CD34
cells/kg, this patient developed a severe pancytopenia, which required
an additional dose of 2 × 106 CD34 cells/kg. After
hemopoietic recovery, myeloid chimerism conversion to donor type was
observed at day +50 following the second DLI, whereas
p190BCR-ABL and p210BCR-ABL RT-PCR converted to
negative at +90 and +120 days, respectively.
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| Fig 3.
Molecular follow-up of patient UPN 251 relapsing after
BMT and responding to DLI.
(A) White blood cell counts, lineage-specific chimerism, and MRD
(p210BCR-ABL and p190BCR-ABL) are depicted.
Numbers express the percentage of host-type hemopoiesis; cytogenetic
relapse (C) and hematological relapse (H) are noted in the figure. (B)
Schematic representation of CML kinetic at molecular level and clinical
parameters.
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Clinical results
At a median follow-up of 34 months (range: 12.5-107 months), 14 of
the 55 patients had developed cytogenetic and clinical relapse. To
evaluate interactions between different potential influential factors
of relapse, we used an analysis that included clinical and molecular
parameters as time-dependent covariates (Table
3).
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Table 3.
Proportional hazards general linear model of potential
influential factor of cytogenetic relapse of CML after BMT
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We detected no significant association of cytogenetic relapse with
either patient age, the time interval between diagnosis and BMT, the
clinical phase at BMT, or the type of immunosuppressive treatment.
Acute and chronic GVHD and T-cell depletion seem to interfere with
disease recurrence. In addition, a significant association between
cytogenetic relapse and BCR-ABL expression or myeloid and T-cell MC was
observed by univariate analysis (Figure 4).
However, multivariate analysis confirmed a positive
p190BCR-ABL as the only independent variable
(P = .002, Table 3). After adjustment for the influence of a
positive p190BCR-ABL, no other factor had a significant
influence on the occurrence of relapse. Detection of MC in myeloid
fraction only presented a trend toward significance
(P = .082). This later event could be justified by the
exceptional relapse observed in donor cells (as in patient
UPN 286).
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| Fig 4.
Actuarial probability of surviving in cytogenetic
remission.
(A) The 9-year actuarial probability of surviving in cytogenetic
remission for patients with positive p190BCR-ABL or
negative P190BCR-ABL. (B) Actuarial probability of
surviving in cytogenetic remission for patients with myeloid MC versus
patients with myeloid DC.
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Interestingly, neither T-cell MC nor p210BCR-ABL expression
was an independent predictor of relapse. Of the 24 patients who showed p210BCR-ABL positivity, 10 cases did not develop either
cytogenetic relapse or p190BCR-ABL expression. The
influence of clinical characteristics on relapse in these patients is
summarized in Table 4. Development of
chronic GVHD was independently associated with
p190BCR-ABL negativity (P = .034) and also
with a better leukemia-free survival in this group of
p210BCR-ABL positive patients.
| |
Discussion |
We report in this study a consistent sequence of molecular findings
that closely parallels disease evolution after allogeneic BMT for CML.
Of the several markers used here for monitoring patient outcome,
chimerism assessment in myeloid cells and p190BCR-ABL mRNA
detection were the best prognostic indicators. Besides allowing a novel
strategy for early identification of CML relapse after BMT, our study
provides new insights into the kinetics of leukemia recurrence and
response to adoptive immunotherapy after relapse in this setting.
Longitudinal monitoring studies of CML patients following BMT were
completed using p210BCR-ABL RT-PCR and chimerism
assessment. Although p210BCR-ABL mRNA positivity,
particularly if detected 6 months post-BMT, has been associated with
subsequent relapse,10 this correlation is not absolute; in
several studies11,29 patients have remained positive in
sustained remission. In the present series, only 14 of 24 patients who
tested positive for p210BCR-ABL after 6 months post-BMT
have undergone relapse. While recently developed quantitative PCR
assays for p210BCR-ABL seem to provide a more accurate
prediction of disease outcome,13,14,30,31 these techniques
are quite laborious and are not suitable for routine clinical work in
most transplantation centers.
In contrast to p210BCR-ABL mRNA, p190BCR-ABL
emerges from our study as a novel marker of CML evolution after BMT. In
fact, p190BCR-ABL positivity by nonquantitative RT-PCR was
associated with impending cytogenetic relapse in the majority of
patients. Moreover, p190BCR-ABL mRNA was not detected in
any patient as a reversible finding nor was it ever found in long-term
survivors. Recently, Lichty et al32 reported that
p190BCR-ABL mRNA is more likely coexpressed, together with
the p210BCR-ABL transcript, in CML patients with high white
blood cell and blast counts as well as in advanced disease. In keeping
with these findings, Carlo-Stella et al33 found that
p190BCR-ABL expression is consistently detectable in the
CML CD34+ cell compartment and progressively decreased in
differentiating elements, and mature cells probably reflect a
differentiation-linked reduced transcription. Finally, the
p190BCR-ABL protein is known to be more strongly
transforming than the p210BCR-ABL protein.34
Therefore p190BCR-ABL detection after transplant could be
not only a marker of total tumor burden but also a more aggressive
feature of the CML clone.
On the other hand, chimerism studies to identify donor versus recipient
hemopoiesis following BMT have been hampered by the use of whole blood
instead of lineage-specific hemopoiesis. This latter issue is
particularly relevant in CML patients after allogeneic BMT. In fact,
the disease that is predominantly expressed in the myeloid compartment
and T lymphocytes rarely belongs to the leukemic clone. Moreover, T
cells frequently survive the conditioning regimen and, therefore, could
affect interpretation of the chimerism findings concerning their
prognostic impact. Besides, analysis of separated cell populations,
especially of antileukemic effector cells (T and NK cells), has not
been studied thoroughly. Although by using quite sophisticated
techniques, such as chromosome-specific fluorescent in
situ hybridization with simultaneous immunophenotyping of interphase cells (FICTION),35 close detection of tumoral kinetic after DLI has recently been reported, the issue of lineage-specific chimerism
evolution and its relationship with MRD and relapse using a simpler
molecular technique remains a matter of controversy.
Mackinnon et al,36 studying a group of T-cell-depleted
patients who had BMTs, found a high incidence of T-cell MC and
subsequent relapse, which suggests that T-cell MC could induce immune
host and/or donor tolerance and thereby abrogate the
graft-versus-leukemia (GVL) effect. However, we and
others37 have detected full DC in T lymphocytes in patients
who relapse, meaning that they do not mediate an efficient immune
effect toward leukemic cells. This immune escape might be due to marked
alterations in expression of surface costimulator molecules and
might lead to anergy or to a lack of alloreactivity in donor
lymphocytes grown in a donor environment.38,39
An important finding of our series is that the presence of T-cell MC
fails to clearly identify subsequent relapses. All 3 patients who
exhibited a transient MC in this subset remain in cytogenetic remission, and 4 patients had full DC T
lymphocytes at the moment of relapse and thereafter. In contrast, when
myeloid cells were studied, we found that all MC patients who expressed recipient type CD15+ cells did not convert to donor origin, and all
relapsed. This makes biological sense when considering the predominant
expression of CML on myeloid series and its susceptibility to be
destroyed with the myeloablative conditioning regimen. This is also in
agreement with van Leeuwen et al,40 who suggest that the
identification of persistent host cells only within the original leukemia lineage can be associated with leukemia relapse. The regrowth
of a tumoral burden, as a consequence of an inefficient immune
surveillance, will be easily detected and found first in the myeloid
compartment. In our experience, when this pattern is established, it
signifies a point of no return, and progressively, B lymphocytes, NK
cells, and even T cells can evolve to recipient origin. We suggest that
the evolution of relapse after BMT is not too different from the
development at the onset of CML, where only myeloid and erythroid
lineages are invariably derived from the leukemic clone.
In our study the consistent kinetic pattern observed in 2 patients was
inversely reproduced after successful DLI was given for relapse. The
disappearance of recipient type CD15+ cells and the negativity of
p190BCR-ABL were the first indications of this change.
Bearing in mind that the expression of p190BCR-ABL can be
interpreted as an indication of the expansion capacity of the
Ph1-positive stem cell compartment,33 these initial
molecular events could be in agreement with the recent
description of Ph1-positive CD34+ cells as the target of
the alloimmune response.41 Moreover, this latter hypothesis
also seems to be sustained by the disappearance of
p190BCR-ABL, which occurred in one patient (UPN
251) when 60% of the host-type granulocytes were still present. Both
patients ultimately converted to RT-PCR negative for the
p210BCR-ABL transcript and remained in hematological remission.
In conclusion, we provide new insights into the biology of the CML
evolution after BMT and suggest a molecular monitoring strategy
suitable for routine work, which may be used to better address clinical
decisions in the post-BMT outcome. For example, patients in hematologic
remission who convert to a positive p210BCR-ABL might be
periodically tested for whole blood chimerism and, whenever increasing amounts of MC are observed, further tested for
myeloid-specific chimerism. Detection of MC and particularly
p190BCR-ABL mRNA in granulocytes infers impending
cytogenetic relapse and might allow early administration of
salvage treatment.
| |
Acknowledgments |
We are particularly grateful to Dr F. Lo Coco for his interest in our
work and his valuable suggestions. We also thank Dr G. Cimino, Dr G. Saglio, and Dr W. Arcese for helpful discussions and for the critical
reading of this manuscript.
| |
Footnotes |
Submitted June 21, 1999; accepted December 16, 1999.
Supported by grant 99/1151 from Fondo Investigacion Sanitario,
Madrid, Spain; Diputacion Provincial de Córdoba,
Córdoba, Spain; Fundación Carlos Haya, Spain; and grant
FIJC-98/ESP-GLAXO (J.S.) from the International Foundation of Jose Carreras.
J.R. and J.S. contributed equally to this work.
Reprints: Josefina Serrano, Hematology Department, University
Hospital "Reina Sofía" Avda. Menendez Pidal s/n. 14004 Córdoba, Spain; e-mail: josefina.serrano{at}iname.com.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
References |
1.
Savage DG, Goldman JM.
Allografting for chronic myeloid leukemia.
Cur Opin in Hemat.
1997;4:369[Medline]
[Order article via Infotrieve].
2.
Thomas DE, Clift RA.
Indications for marrow transplantation in chronic myelogenous leukemia.
Blood.
1989;73:861[Free Full Text].
3.
Kolb HJ, Schatteeberg A, Goldman JM, et al.
Graft-versus-leukemia effect of donor lymphocyte transfusions in marrow grafted patients.
Blood.
1995;86:2041[Abstract/Free Full Text].
4. Collins RH, Shpilberg O, Drobyski WR, et al. Donor leukocyte infusions
in 140 patients with relapsed malignancy after allogeneic bone marrow
transplantation. J Clin Oncol. 197;15:433.
5.
Higano CS, Chielens D, Raskind W, et al.
Use of -2a-interferon to treat cytogenetic relapse of chronic myeloid leukemia after marrow transplantation.
Blood.
1997;90:2549[Abstract/Free Full Text].
6.
Arcese W, Mauro FR, Screni M, et al.
Interferon therapy for Ph1 positive CML patients relapsing after T cell-depleted allogeneic bone marrow transplantation.
Bone Marrow Transplant.
1990;5:309[Medline]
[Order article via Infotrieve].
7.
Mackinnon S, Papadopoulus EB, Carabasi MH, et al.
Adoptive immunotherapy evaluating escalating doses of donor leukocytes for relapse of chronic myeloid leukemia after bone marrow transplantation: separation of graft-versus-leukemia from graft-versus-host disease.
Blood.
1995;86:1261[Abstract/Free Full Text].
8.
Rapanotti MC, Arcese W, Buffolino S, et al.
Sequential molecular monitoring of chimerism in chronic myeloid leukemia patients receiving donor lymphocyte transfusions for relapse after bone marrow transplantation.
Bone Marrow Transplant.
1997;19:703[Medline]
[Order article via Infotrieve].
9.
Bilhou-Nabera C, Bernard P, Marit G, et al.
Serial cytogenetic studies in allografted patients with chronic myeloid leukemia.
Bone Marrow Transplant.
1992;9:263[Medline]
[Order article via Infotrieve].
10.
Miyamura K, Barrett AJ, Kodera Y, Saito H.
Minimal residual disease after bone marrow transplantation for chronic myeloid leukemia and implications for graft versus leukemia effect: a review of recent results.
Bone Marrow Transplant.
1994;14:201[Medline]
[Order article via Infotrieve].
11.
Radich JP, Gehly G, Gooley T, et al.
Polymerase chain-reaction detection of the bcr-abl fusion transcript after allogeneic marrow transplantation for chronic myeloid leukemia: results and implications in 346 patients.
Blood.
1995;85:2632[Abstract/Free Full Text].
12.
Román J, Martín C, Torres A, et al.
Importance of mixed chimerism to predict relapse in persistently bcr-abl long survivors after bone marrow transplantation for chronic myelogenous leukemia.
Leuk Lymphoma.
1998;28:541[Medline]
[Order article via Infotrieve].
13.
Gaiger A, Lion T, Kalhs P.
Frequent detection of bcr-abl specific mRNA in patients with chronic myeloid leukemia (CML) following allogeneic and syngeneic bone marrow transplantation.
Leukemia.
1993;7:1766[Medline]
[Order article via Infotrieve].
14.
Cross NCP.
Quantitative PCR techniques and applications.
Br J Haematol.
1995;89:693[Medline]
[Order article via Infotrieve].
15.
Lin F, van Rhee F, Goldman JH, Cross NCP.
Kinetics of increasing BCR-ABL transcripts number in chronic myelogenous leukemia patients who relapse after bone marrow transplantation.
Blood.
1996;87:4473[Abstract/Free Full Text].
16.
Serrano J, Roman J, Herrera C, et al.
Increasing mixed haematopoietic chimaerism after BMT with total depletion of CD4+ and partial depletion of CD8+ lymphocytes is associated with a higher incidence of relapse.
Bone Marrow Transplant.
1999;23:475[Medline]
[Order article via Infotrieve].
17.
Gardiner N, Lawler M, O'Riordan J, Duggan C, De Arce M, McCann SR.
Persistent donor chimaerism is consistent with disease-free survival following BMT for chronic myeloid leukaemia.
Bone Marrow Transplant.
1997;20:235[Medline]
[Order article via Infotrieve].
18.
Elmmagacli AH, Becks HW, Beelen DW, et al.
Detection of minimal residual disease and persistence of host type hematopoiesis: a study in 28 patients after sex mismatched, non-T cell-depleted allogeneic bone marrow transplantation for Philadelphia positive chronic myeloid leukemia.
Bone Marrow Transplant.
1995;16:823[Medline]
[Order article via Infotrieve].
19.
Van Rhee F, Hochhaus A, Lin F, Melo JV, Goldman JM, Cross NCP.
P190 BCR-ABL mRNA is expressed at low levels in p210-positive chronic myeloid and acute lymphoblastic leukemias.
Blood.
1996;87:5213[Abstract/Free Full Text].
20.
Saglio G, Pane F, Gottardi E, et al.
Consistent amounts of acute leukemia-associated p190 BCR/ABL transcript are expressed by chronic myelogenous leukemia patients at diagnosis.
Blood.
1996;87:1075[Abstract/Free Full Text].
21.
Herrera C, Torres A, Garcia-Castellano JM, et al.
Prevention of graft-versus-host disease in high risk patients by depletion of CD4+ and reduction of CD8+ lymphocytes in the marrow graft.
Bone Marrow Transplant.
1999;23:443[Medline]
[Order article via Infotrieve].
22.
Roman J, Andres P, Alvarez MA, Torres A.
Salting out procedure for isolation of DNA from stored bone marrow slides for PCR [letter].
Eur J Haematol.
1993;50:237[Medline]
[Order article via Infotrieve].
23.
Chomczynski P, Sacchi N.
Single-step method of RNA isolation by acid guanidinium thyocyanate-phenol-chloroform extraction.
Anal Biochem.
1987;162:156[Medline]
[Order article via Infotrieve].
24.
Kwok S, Higuchi R.
Avoiding false positives with PCR.
Nature.
1989;339:237[Medline]
[Order article via Infotrieve].
25.
Miller SA, Dykes DD, Polesky HF.
A simple salting out procedure for extracting DNA from human nucleated cells [letter].
Nucleic Acids Res.
1988;16:1215[Free Full Text].
26.
Roman J, Garcia MJ, Serrano J, et al.
Chimerism analysis in long-term survivor patients after bone marrow transplantation for severe aplastic anemia.
Haematologica.
1997;82:588[Abstract/Free Full Text].
27.
Kaplan EL, Meier P.
Non-parametric estimation from incomplete observations.
J Am Stat Assoc.
1958;53:457.
28.
Cox DR.
Regression models and life-tables.
J R Stat Soc.
1972;34:187.
29.
Miyamura K, Tahara T, Tanimoto M, et al.
Long persistent bcr-abl positive transcript detected by polymerase chain reaction after bone marrow transplant for chronic myelogenous leukemia without clinical relapse: a study of 64 patients.
Blood.
1993;81:1089[Abstract/Free Full Text].
30.
Cross NCP, Feng L, Chase A, Bungey J, Hughes TP, Goldman JM.
Competitive polymerase chain reaction to estimate the number of BCR-ABL transcripts in chronic myeloid leukemia patients after bone marrow transplantation.
Blood.
1993;82:1929[Abstract/Free Full Text].
31.
Lion T, Henn T, Gaiger A, Mor W, Gadner H.
Early detection of relapse after bone marrow transplantation in patients with chronic myelogenous leukemia.
Lancet.
1993;341:275[Medline]
[Order article via Infotrieve].
32.
Lichty BD, Keating A, Callum J, et al.
Expression of p210 and p190 bcr-abl due to alternative splicing in chronic myelogenous leukaemia.
Br J Haematol.
1998;103:711[Medline]
[Order article via Infotrieve].
33.
Carlo-Stella C, Savoldo B, Sammarelli G, et al.
Detection of p190 and p210 bcr-abl mRNA in CML-derived CD34+, CD34+ and CD15+ cells.
Blood.
1998;92:128b.
34.
Voncken JW, Kaartinen V, Pattengale PK, Germerrad WT, Groffen J, Heisterkamp N.
BCR/ABL P210 and P190 cause distinct leukemia in transgenic mice.
Blood.
1995;86:4603[Abstract/Free Full Text].
35.
Baurmann H, Nagel S, Binder T, Neubauer A, Siegert W, Huhn D.
Kinetics of the graft-versus-leukemia response after donor leukocyte infusions for relapsed chronic myeloid leukemia after allogeneic bone marrow transplantation.
Blood.
1998;92:3582[Abstract/Free Full Text].
36.
Mackinnon S, Barnett L, Heller G, O'Reilly J.
Minimal residual disease is more common in patients who have mixed T-cell chimerism after bone marrow transplantation for chronic myelogenous leukemia.
Blood.
1994;83:3409[Abstract/Free Full Text].
37.
Verdonck LF, Dekker AW, Gijsbert C, van Kempen ML, Lokhorst HM, Nieuwenhuis HK.
Allogeneic bone marrow transplantation with a fixed low number of T cells in the marrow graft.
Blood.
1994;83:3090[Abstract/Free Full Text].
38.
Dermine S, Mavroudis D, Jiang Y-Z, Hensel N, Molldrem J, Barret AJ.
Immune escape from a graft-versus-leukemia effect may play a role in the relapse of myeloid leukemias following allogeneic bone marrow transplantation.
Bone Marrow Transplant.
1997;19:989[Medline]
[Order article via Infotrieve].
39.
Guinen EC, Gribben JG, Boussiotis VA, Freeman GJ, Nadler LM.
Pivotal role of the B7:CD28 pathway in transplantation tolerance and tumor immunity.
Blood.
1994;84:3261[Abstract/Free Full Text].
40.
van Leeuwen JEM, van Tol MJD, Joosten AM, Wijnen, TJh, Hhan PM, Vossen JM.
Mixed T-lymphoid chimerism after allogeneic bone marrow transplantation for hematologic malignancies of children is not correlated with relapse.
Blood.
1993;82:1921[Abstract/Free Full Text].
41.
Smit WM, Rijnbeek M, van Bergen CAM, Fibbe WE, Willenze R, Falkenburg JH.
T cells recognizing leukemic CD34+ progenitor cells mediate the antileukemic effect of donor lymphocyte infusions for relapsed chronic myeloid leukemia after allogeneic stem cell transplantation.
Proc Natl Acad Sci U S A.
1998;95:10,152[Abstract/Free Full Text].
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Abstract
Introduction