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Detection of Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia
by Polymerase Chain Reaction: Possible Eradication of Minimal Residual
Disease by Marrow Transplantation
By K. Miyamura, M. Tanimoto, Y. Morishima, K. Horibe, K. Yamamoto, M. Akatsuka, Y. Kodera, S. Kojima, K. Matsuyama,
N. Hirabayashi, M. Yazaki, K. Imai, Y. Onozawa, A. Kanamaru, S.Mizutani, and H. Saito
Minimal residual disease (MRD) in patients with Philadelphia
chromosome-positive acute lymphoblastic leukemia (Phl
ALL) who received allogeneic (n = 9) or autologous (n = 6)
bone marrow transplantation (BMT) was evaluated by the
polymerase chain reaction (PCR) for the bcr-ab1 transcript.
Twelve patients received BMT at the time of hematologic and
cytogenetic remission. However, MRD was detected in 8 of
10 patients evaluated. Seven patients, including three who
had MRD before BMT, continue to have a disease-free
survival 5 to 64 months after BMT. Twenty-one specimens
obtained from these patients at various times after BMT did
not show MRD. In three patients, MRD detected just before
BMT seems to be eradicated by BMT protocol. The other
eight patients developed cytogenetic or hematologic relapses 2 to 8 months after BMT. Seven of 14 samples from
these patients demonstrated MRD, which preceded clinical
relapse by 3 to 9 weeks. Thus, this technique for the
detection of MRD appears to be useful for the more precise
assessment of various antileukemia therapies and for early
detection of leukemia recurrence.
o 1992 by TheAmerican Society of Hematology.
B
some occurs in most cases of chronic myelogenous leukemia
(CML) and in 10% to 30% of ALL cases, and results from a
reciprocal translocation between chromosome 9q and 22q.
This translocation involves the movement of the abl protooncogene on chromosome 9 to the breakpoint cluster
region (bcr) gene on chromosome 22.4 This results in
transcript of an 8.5-kb bcr-ab1 mRNA, which encodes a
hybrid protein of 210 Kd (~210): Most of Phl ALL cases
have a translocation site different from that of CML, and
more 5’ bcr exon (BCR) is used in the formation of the
fused bcr-abl gene.6This results in a smaller bcr-abl mRNA
of 7 kb, encoding a 190-Kd protein ( ~ 1 9 0 )To
. ~ date, there
have been several studies reporting that residual bcr-ab1
transcripts in CML can be detected by this technique after
BMT.8-”However, the clinical significance of the residual
bcr-abl-positive clones after BMT is still controversial.
In Phl ALL, a report from our group suggested that
residual bcr-abl transcript was directly correlated with
hematologic relapse, although the number of patients
studied was small.” Here we extend our findings and report
the clinical details and the results of PCR on 15 Phl ALL
patients. In addition, we assessed their remission status
before and after BMT more precisely. In some of these
patients, MRD detectable after chemotherapy seems to be
eradicated by BMT.
ONE MARROW transplantation (BMT) has been
performed as one mode of cure-oriented therapy for
high-risk acute lymphoblastic leukemia (ALL), such as
Philadelphia chromosome-positive (Phl) ALL.’,’ Nevertheless, recurrence of the original disease remains one of the
major obstacles for obtaining long-term survivals after
BMT. Leukemia recurrence has been considered to originate from minimal residual disease (MRD) that escaped
intensive chemoradiotherapy or posttransplant immune
surveillance. As most patients receive BMT at the hematologic remission phase, it is not clear whether some of the
recipients might have been cured by conventional chemotherapy. For such patients, BMT might be only hazardous.
Therefore, more sensitive techniques for the detection of
MRD, which cannot be diagnosed by conventional hematologic or cytogenetic analysis, may provide insight into
improving the treatment strategy.
Recently, polymerase chain reaction (PCR) has become
available for the detection of low levels of the chimeric
bcr-abl transcripts in Phl leukemia patients.’ Phl chromo-
From the First Department of Intemal Medicine and Pediabics,
Nagoya University School of Medicine; the Department of Intemal
Medicine and Pediatrics, Japanese Red Cross Nagoya First Hospital;
the Department of Intemal Medicine, Japanese Red Cross Nagoya
Second Hospital; the Department of Pediatrics, Nagoya City University, Nagoya; the Department of Chemotherapy and Intemal Medicine,
Tokyo Metropolitan Komagome Hospital; the Second Department of
Intemal Medicine, Hyogo Medical School, Hyogo; and the Department of Krology, National Children’s Medical Research Center,
Tokyo, Japan.
Submitted August 5, 1991; accepted October 24, 1991.
Supported by Grants-in-Aidfrom the Ministry of Education, Science
and Culture; and from the Minishy of Health and Welfare in Japan
and by Aichi Blood Disease Research Foundation.
Address reprint requests to Koichi Mjamura, MD, First Department
of Intemal Medicine, Nagoya University School of Medicine, 65
Tsurumai-cho, Showa-ku, Nagoya 466, Japan.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1992 by The American Society of Hematology.
0006-4971 f92/7905-OO06$3.OO/0
1366
PATIENTS AND METHODS
The characteristics of 15 Phl ALL patients are given in Table 1.
All had Phl chromosome at the time of diagnosis. The clinical
diagnosis of Phl ALL was made according to the standard clinical
findings (acute onset of leukemia, leukemia cells with lymphoblastic features, no cytogenetic abnormalities in the remission marrow,
etc). Nine of 15 patients received allogeneic BMT, while the other
six patients received autologous BMT because of the absence of
suitable donors. In these six patients, five patients (cases 1, 9, 10,
13, and 15) with common ALL antigen (CALLA)-positive leukemia cells received their bone marrow that had been purged with
mixture of three distinct CALLA monoclonal antibodies and baby
rabbit complement as described previously.” Case 2, whose leukemia cells were negative for CALLA antigen, received autologous
marrow without purging. Preparative regimens for BMT are also
shown in Table 1. Details of the BMT regimens have been reported
previ~usly.”.’~
When the karyotypes of more than 20 metaphase
cells were all normal, the patients were judged to be in cytogenetic
Blood, Vol79, No 5 (March 1). 1992: pp 1366-1370
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PCR ANALYSIS OF TRANSPLANTED Phl ALL
1367
Table 1. Characteristicsof 15 Patients With Phl ALL
Case
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
AgelSex
lmmunophenotype
4/ F
CALLA
6/F
22/F
39/F
Pre-B
Pre-B
21/M
22/F
19/F
8/M
4/F
6/F
7/M
20/M
52/F
13/M
39/M
CALLA
CALLA
CALLA
Pre-B
CALLA
CALLA
CALLA
CALLA
Pre-B
CALLA
CALLA
CALLA
Status at BMT
(cytogeneticstudies,
Phllmetaphases)
2CR (0/20)
1CR (0/20)
1REL (3/20)
ICR (0/20)
PREL (8/20)
3REL (20/20)
2CR (0120)
2CR (0/20)
1CR (0/20)
1CR (0/20)
3CR (0/20)
ICR
ICR
ICR
ICR
(0/20)
(0/20)
(0/20)
(0/20)
BMT
Preparatory Regimen
Results of PCR
at Initial
Diagnosis
Autologous*
Autologous
Allogeneic
Allogeneic
Allogeneic
Allogeneic
Allogeneic
Allogeneic
Autologous*
Autologous*
Allogeneic
Allogeneic
Autologous*
Allogeneic
Autologous*
L-PAM + TBI
L-PAM + BU
CA CY + TBI
CA + CY + TBI
CA + CY + TBI
CY BU
CA + CY TBI
CA + CY + TBI
L-PAM + BU
L-PAM + TBI
CA + CY + TBI
CA + CY + TBI
CA + CY + TBI
CA + CY + TBI
CA + CY + TBI
BCR-ab12
BCR-ab12
BCR-ab12
BCR-ab12
BCR-ab12
BCR-ab12
BCR-ab12
bcr3-ab12
BCR-ab12
BCR-ab12
BCR-ab12
bcr3-ab12
BCR-ab12
BCR-ab12
bcr2-abi2
+
+
+
Abbreviations: CR, complete remission; REL, relapse; CA, cytosine arabinoside (8 g/m2);CY, cyclophosphamide (120 mg/kg); TBI, total body
irradiation (10-12Gy,fractionation); BU, busulfan (16 mglkg);L-PAM, melphalan (120mglm’).
*Purgingwith anti-CALLA antibodies.
remission. All patients were in cytogenetic and hematologic remission at the time of the PCR study.
Thirteen patients (cases 1 through 13) were prospectively
studied. After informed consent was obtained, fresh bone marrow
samples were collected just before BMT and 1, 2, 3, 6, and 12
months after BMT. The other two patients (cases 14 and 15) were
studied several yews after BMT. In addition, frozen leukemia cells
at the time of initial diagnosis were used. Phl-positive cell lines K
562 and MR26 (kindly provided by Dr J. Okamura, Kyuushu
Cancer Center, Fukuoka, Japan), which express the p210 or
pl90-type transcripts, respectively, were used as positive controls
in PCR after appropriate dilution. RNA extracted from the
peripheral blood mononuclear cells of healthy volunteers’ material
was used as a negative control.
Oligonucleotides used in this study were synthesized by an
Applied Biosystems 391A DNA synthesizer (Applied Biosystems,
Foster City, CA). Many sets of primers were used for detection of
fused transcripts: bcr2 (exon 2)-abZ2 and bcr2-abZ3 (for p210 type
transcripts), and BCR (more 5’ bcr exon)-abZ2 and BCR-@bZ3(for
p190 type transcript).Sequencesof mainly used primers and junctional
probes for Squthern analysis were as follows: primer “abZ-2” (abZ
exon 2), 5‘-GCTGAAAGTCAGATGCTATC-3’;
primer “bcr”
primer “BCR’
(bcr exon 2), 5’-ATTCCGTGACCATCAATAA-3’;
(more 5’ bcr exon), 5‘-GlTGTCGTGTCCGAGGCCAC-3’;and
junctional probes “bcr2-abZ2”: 5’-GCTGAAGGGCTTCTTCCTTATT GATG-3’, “bcr3-abZ2,” 5’-GCTGAAGGGCTTTTGAACTCTGCTTA-3’; and “BCR-abZ2,” 5’-GCTGAAGGGCTTCTGCOTCTCCAT-3’.To confirm the quality of RNA, amplification
of B2-microglobulin was used with primer “BM1” (exon 1):
5’-TCTCGCTCCTGGCCGCT-3’, and primer ‘‘BM2” (exon
2): 5’-CCCACTTAACTATCTTGGGT-3’.
Total RNA was prepared by the acid guanidine thiocyanate/
phenol/chloroform method. Reverse transcription and PCR amplification was performed according to the previously described
methods.” PCR was performed for 35 cycles using a Perkin Elmer
Cetus DNA Thermal Cycler (Emeryville, CA) (denaturation at
94°C for 1 minute, annealing at 55°C for 1 minute, and then
extension at 72°C for 2 minutes). For detection of MRD, PCR
productswere analyzed by Southern hybridizationwith a junctional
probe that had been 5’ end-labeled with ”P-ATP. Details of this
procedure was also described previo~sly.’~
Using this approach, we could detect a single Phl-positive cell in
lo5normal cells. Precautions to prevent the cross-contamination of
the amplified material were taken according to the recommendations of Kwok and Higuchi.“ All experiments were first performed
with negative controls at all steps of the experiment to ensure the
absence of cross-contamination. If all results were negative, the
same procedure was repeated with a diluted positive control.
RESULTS
To confirm the existence of bcr-ab1 transcripts, frozen
cells from all patients stored at initial diagnosis were
examined by PCR. Twelve patients were found to have p190
type transcript (BCR-abl2, 128 bp) and the other three
patients (cases 8, 12, and 15) had p210 type expression
(bcr2-ab12, 61 bp in one patient and bcr3-ab12, 136 bp in
two) (Table 1).
Results of clinical course and PCR analysis are shown in
Fig 1. Twelve patients were proved to be in complete
remission by standard hematologic and cytogenetic criteria
just before BMT. Ten patients in remission were examined
for the presence of MRD using fresh bone marrow cells.
Fused bcr-ab1 trafiscript was detected in eight patients (Fig
2, A and C).
Seven patients (cases 9 through 15) are alive and in
complete remission 5 to 64 months after BMT. Six of these
patients received BMT during their first remission and one
in his third remission; however, three patients (cases 10,11,
and 13) had MRD just before BMT. After BMT, bcr-ab1
transcripts were not detected by PCR in 21 samples
obtained from these seven disease-free survivors (Fig 1).
The other eight patients (cases 1 through 8) suffered
hematologic or cytogenetic relapses (clinical relapse) 2 to 8
months after BMT, and they died 4 to 14 months after their
BMT. All but two (cases 2 and 4) of these patients were
negative for MRD by PCR analysis shortly after BMT.
However, five patients (cases 1, 3, 5, 6, and 7) eventually
expressed bcr-ab1 transcript detected by PCR (molecular
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MIYAMURA ET AL
1368
BMT
U"
Table 2. PCR Results and Clinical Relapse in 13 Patients
Prospectively Studied
1
1
2
Results of PCR
1
Nega t iv e
Positive
4
Clinical Relapse
S
(I
l
1
+
717
7/26
P c .01'
I
*Fisher's exact test.
9
10
11
tween 1 and 12 months after BMT had significant correlation with clinical relapse (Tablc 2).
12
13
14
DISCUSSION
15
BMT
3M
6M
In the present study, we have used PCR as a tool for
monitoring MRD in Phl ALL patients who received BMT.
Most paticnts who had achievcd cytogenetic and hematologic remission after conventional chemotherapy still had
hcr-ahl-positive clones by PCR analysis. If this is an
indicator for future clinical relapse, conventional chemotherapy appears to be inadequate to eradicate all of the
leukemic cells and to achieve a durable remission in most
patients. Among 11 patients who still had a Phl clone by
cytogenetic or PCR analysis just before BMT, nine were
negative for the hcr-ah1 transcripts shortly after BMT.
Thercfore, our BMT regimen was able to reduce the level
of leukemic cells to less than 1 in 10' (our PCR technique
limit). Six patients have disease-free survival of more than
12 months without thc sign of molecular relapse. They are
thought to be cured by BMT because all clinical relapses
occurred within 8 months after BMT in this study. In three
patients (cases 10, 11, and 13), MRD detected by PCR
before BMT seems to be eradicated by transplant procedures. These data support the current concept that Phl
12M
Fig 1. Clinical course and PCR results in the 15 P h l ALL patients.
(O), PCR-positive; (0).PCR-negative; (W), presence of leukemia
cytogenetically or hematologically; (Z),
absence of leukemia cytogenetically and hematologically. M, months; ND, not done; t, dead.
relapse) (Fig 2B). Median interval between PCR positivity
and clinical relapse was 4 weeks. One paticnt (case 8)
relapsed hematologically without a prior positive PCR
result (Figs 1 and 2C). To eliminate the possibility of
false-negative result, amplification of P2microglobulin was
used. All samples demonstrated correct amplification, confirming the presence of intact RNA (Fig 2, B and C).
In 13 patients (cases 1 through 13) studicd prospectively,
the value of PCR results to prcdict relapse was examined.
Of 33 PCR analyses, 7 were positive and 26 were negative.
PCR positivity was well related with clinical relapsc in the
near future. Conversely, negative results did not always
mean cure of disease. Seven negative PCR analysis were
eventually related with clinical relapse. PCR rcsults be-
A
N 1 2 4 N 7 910N1113
"..*-. *-
-
128
136-
1 1 ' 2 3 3 "
-
8
I
128-
B
C
4 55'5"66"77"
8
Fig 2. Detection of bcr-8bl and B2-microglobulin
transcripts by PCR amplification pre-BMT and postBMT. Lane numbers refer t o cases in Table 1. Lane N,
peripheral blood from normal volunteer. (A) p190
type transcript (BCR-8blZ 128 bp) just before BMT.
The panel shows an autoradiogram of Southern blot
probed with junctional probe "BC/?-8b/?'. Amplified
product is visible in cases 1,2,4,7,10, 11, and 13. (6)
PCR results of patients (cases 1through 7, p190 type)
who eventually relapsed are shown. The top panel is
the result of hybridization with p190-specific probe.
Cases 1, 3,5, 6, and 7 show negative results at early
posttransplant phase, but turned out t o be positive
PCR in subsequent studies. The bottom panel shows
ethidium bromide staining. A band corresponding t o
the 340-bp PCR product of B2-microglobulin is shown
in all lanes, which confirms that intact RNA was
present. (C) PCR result of case 8. Upper panel shows
p210 type transcript (136 bp) hybridized with junctional probe "Lcr3-8b/?'. bcr-8bl RNA detected before BMT (lane 8) is not shown 3 months after BMT
(lane 8'). Lower panel presents the amplification of
62-microglobulin.
From www.bloodjournal.org by guest on July 28, 2017. For personal use only.
PCR ANALYSIS OF TRANSPLANTED Phl ALL
1369
ALL patients belong to a high-risk group and that BMT
should be considered as the first-line therapy for them."
Eight patients had clinical relapse 2 to 8 months after
BMT. However, six of these eight patients had been
PCR-negative shortly after BMT. In these patients, amount
of minimal residual leukemia cells were less than the
detectable level of PCR during the early phase of postBMT. Among 35 tests after BMT, only seven samples from
seven patients of this group demonstrated PCR positivity.
Detection of the bcr-ab1 transcripts by PCR preceded
clinical relapse by 3 to 9 weeks. This would indicate that
molecular relapse after BMT correlates with clinical relapse in Phl ALL, as we have previously suggested."
Furthermore, five patients were initially negative by PCR
analysis but turned positive 1 to 3 months later. Thus,
leukemia cells in Phl ALL patients appear to have highly
proliferative capacity.
It is interesting to note that the significance of positive
bcr-ab1 transcripts by PCR after BMT in CML patients
appears to be different from that in Phl ALL patients.
Despite persistent bcr-ab1 expression, positive PCR analysis
is not always an early sign of clinical relapse in CML.*-"
Blood cells from nine patients with CML in the chronic
phase were studied 3 to 6 months after BMT and six were
PCR-positive; three were negative on subsequent studies
and all six patients remain in remission 9 to 18 months after
BMT." These distinct observations in Phl ALL and CML
may be interpreted as follows. The tumor burden in
patients with CML in the chronic phase is much greater
than that of ALL patients in remission state, and some
CML leukemic clones tend to survive beyond preconditioning, which results in MRD detected by PCR. Yet, these
CML clones tend to grow very slowly'8 and, therefore, do
not appear to predict hematologic relapse in the near
future. In contrast, Phl ALL clones may possess high
proliferative potential, and the existence of minimal residual Phl clones correlates with imminent clinical relapse.
Among six patients who received autologous BMT, four
(cases 1, 2, 10, and 13) were proved to have MRD before
BMT. Three patients who received purged bone marrow
continue to have long-term survival whereas the other
patient (case 2) who received unpurged marrow developed
clinical relapse. It would be interesting to know if MRD was
removed by our purging protocol. Further study is needed
to evaluate the efficacy of our bone marrow purging
protocol.
In one report, 10 Phl ALL patients received allogeneic
BMT, and six were alive and well 6 to 30 months (median 19
months) after BMT.19 Four patients died with transplantrelated complications. However, in our study patients died
only from recurrence of leukemia. The reason of these
distinct results is unclear but it may come from, in part, the
difference of BMT protocol and, in part, the difference of
median age of patients studied (19 years in this study v 28
years in the previous report).
ACKNOWLEDGMENT
The authors thank Sayoko Sugiura for technical assistance and
Drs Tohru Tahara and Kohei Kawashima for useful discussion.
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MIYAMURA ET AL
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From www.bloodjournal.org by guest on July 28, 2017. For personal use only.
1992 79: 1366-1370
Detection of Philadelphia chromosome-positive acute lymphoblastic
leukemia by polymerase chain reaction: possible eradication of
minimal residual disease by marrow transplantation
K Miyamura, M Tanimoto, Y Morishima, K Horibe, K Yamamoto, M Akatsuka, Y Kodera, S Kojima,
K Matsuyama and N Hirabayashi
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