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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 3 (February 1), 1998:
pp. 1021-1028
E2A-PBX1 Chimeric Transcript Status at
End of Consolidation Is Not Predictive of Treatment Outcome in
Childhood Acute Lymphoblastic Leukemias With a t(1;19)(q23;p13): A
Pediatric Oncology Group Study
By
Stephen P. Hunger,
Majilinde Z. Fall,
Bruce M. Camitta,
Andrew J. Carroll,
Michael P. Link,
Stephen J. Lauer,
Donald H. Mahoney,
D. Jeanette Pullen,
Jonathan J. Shuster,
C. Philip Steuber, and
Michael L. Cleary
From Section of Pediatric Hematology/Oncology, the Department of
Pediatrics; University of Colorado School of Medicine, Denver;
Section of Pediatric Hematology/Oncology, the Department
of Pediatrics and Laboratory of Experimental Oncology, the Department
of Pathology, Stanford University School of Medicine, CA; Midwest
Children's Cancer Center, Pediatric Hematology/Oncology, Milwaukee,
WI; University of Alabama, Laboratory of Medical Genetics, Birmingham;
Emory University, Pediatric Hematology/Oncology, Atlanta, GA; Baylor
University, the Department of Pediatric Oncology, Houston,
TX; University of Mississippi Medical Center, the Department of
Pediatric Hematology/Oncology, Jackson; and Pediatric Oncology Group
Statistical Office, University of Florida, Gainesville.
 |
ABSTRACT |
A t(1;19)(q23;p13) is detected cytogenetically in approximately 5%
of childhood acute lymphoblastic leukemias (ALLs) and its presence has
been associated with an increased risk of relapse in several
previously-completed Pediatric Oncology Group (POG) clinical trials.
The t(1;19) fuses E2A to PBX1 in more than 95% of
cases and this molecular abnormality can be reliably identified by
polymerase chain reaction (PCR)-mediated amplification of
E2A-PBX1 chimeric mRNAs. We used a nested PCR assay,
which reproducibly detected a 104- to 105-fold
dilution of t(1;19)+ into t(1;19) cells,
to evaluate minimal residual disease (MRD) in 48 children with
t(1;19)+ ALL enrolled in POG clinical trials for lower
(POG 9005) and higher (POG 9006) risk ALL. Peripheral blood (PB) and
bone marrow (BM) samples were collected prospectively at the end of
consolidation (weeks 25 and 31 after end of induction) and the presence
or absence of PCR-detectable MRD was correlated with clinical outcome.
Overall, 41 of 148 (28%) samples were PCR+. Of the 65 time points with informative results from both PB and BM, PCR results
were concordant for 51 pairs (10 PB+/BM+,
41 PB/BM) and discordant for 14 (5 PB+/BM, 9 PB/BM+), indicating that assessment of
only PB or only BM can inaccurately classify some PCR+
cases as PCR. There were no significant differences in
event-free survival between PCR+ and PCR
patients. We conclude that qualitative detection of MRD by
amplification of E2A-PBX1 chimeric mRNAs at the end of
consolidation was not significantly predictive of outcome for children
treated on POG 9005/9006 and that such results should not be used to
alter therapy for patients with t(1;19)+ ALL.
| |
INTRODUCTION |
THE ADVENT OF the polymerase chain
reaction (PCR) has facilitated detection of minimal residual disease
(MRD) in patients with leukemia who are in complete clinical remission
during and after therapy.1 PCR-based approaches typically
can detect 104- to 106-fold dilutions of cells
carrying the specific marker into marker-negative cells, which is
significantly more sensitive than the 1% to 5% threshold of detection
for traditional methodologies.1 Several different molecular
markers can be monitored by PCR, chief among them being
leukemia-specific Ig or T-cell receptor (TCR) gene rearrangements that are unique to each patient, and chimeric mRNAs produced by chromosomal translocations. There are advantages and disadvantages to each strategy, with amplification of Ig/TCR
gene rearrangements being more widely applicable while amplification of
chimeric transcripts has the theoretical advantage of measuring a
marker directly linked to leukemogenesis rather than a functionally neutral genetic alteration. A major goal of this field of research is
to determine if patients can be reliably stratified on the basis of PCR
results into subgroups with differential likelihoods of being cured
with contemporary chemotherapy regimens. Development and
refinement of PCR-based MRD assays potentially offers the opportunity
to "tailor" chemotherapy based on molecular assessment of
treatment response.
The t(1;19)(q23;p13) occurs in 5% to 6% of all childhood acute
lymphoblastic leukemias (ALLs).2 More than 95% of t(1;19)s fuse the chromosome 19 gene E2A to the chromosome 1 gene
PBX1, producing fusion mRNAs that encode for E2A-PBX1 chimeric
proteins.3-5 E2A-PBX1+
t(1;19)+ ALLs are typically classified as pre-B ALLs as
they express cytoplasmic, but not surface, Ig
(cIg+/sIg). Approximately 25% of pre-B
ALLs contain a t(1;19) and E2A-PBX1 fusion.6-9
Genomic breakpoints in E2A-PBX1+
t(1;19)+ ALLs occur in restricted regions of E2A
and PBX1 and join identical portions of E2A and
PBX1 in the processed fusion mRNA.3-5,10 A small
percentage of cases have 27 identical nucleotides of uncertain origin
located at the junction of E2A and PBX1 sequences in
the processed fusion mRNAs.11 Despite this minor
difference, fusion mRNAs can be amplified with a single set of primers
from all cases of E2A-PBX1+ t(1;19)+
ALL molecularly analyzed to date except for a single case with variant
E2A-PBX1 fusion mRNAs.5,11-15 We have previously
shown that PCR can detect MRD in t(1;19)+ ALL patients in
remission who subsequently experienced relapse.5 Others
have also anecdotally described PCR detection of MRD in small cohorts
of t(1;19)+ ALL patients, but the clinical value of this
approach has not been defined.14,16-18
Presence of the t(1;19) has been associated with an inferior treatment
outcome on several Pediatric Oncology Group (POG) and St Jude
Children's Research Hospital (Memphis, TN) clinical
trials and has been shown to largely account for the inferior prognosis observed in pre-B as compared to early pre-B ALLs in these
studies.7-9 Published results from other groups have
generally, but not invariably, demonstrated similar adverse outcomes
for t(1;19)+ ALLs.19,20 Recent trials have
shown that this adverse outcome for t(1;19)+ patients can
be overcome by more intensive therapies.9,20 In 1991, the
POG began a pair of clinical trials for lower (POG 9005) and higher
(POG 9006) risk children with ALL. After a standard 3 (9005) or 4 (9006) drug induction, these trials allocated all t(1;19)+
patients to a single chemotherapy regimen that was identical between
the 9005 and 9006 studies. This study design provided us with the
opportunity to evaluate the predictive value of MRD detection by
amplification of E2A-PBX1 chimeric mRNAs in a large cohort of
similarly treated children with t(1;19)+ ALL.
| |
MATERIALS AND METHODS |
Study group and design.
Patients less than 22 years of age enrolled in POG 9005/9006 in whom a
t(1;19)(q23;p13) was identified cytogenetically at the time of
diagnosis formed the study group. In total, 30 t(1;19)+
patients were enrolled in POG 9005 and 44 in POG 9006. After receiving
a standard three (9005) or four (9006) drug induction (prednisone,
vincristine, L-Asparaginase with or without daunorubicin), t(1;19)+ patients were nonrandomly assigned to an
anti-metabolite-based consolidation and continuation treatment arm
which was identical between the two studies.21 The
protocols called for paired samples of peripheral blood (PB) and bone
marrow (BM) to be collected from t(1;19)+ patients at
defined times during therapy and shipped by overnight courier to a
central laboratory at Stanford University. In the current study, we
analyzed all available PB and BM samples collected at two time
points at the conclusion of consolidation chemotherapy (weeks 25 and 31 after completion of induction). The 9005/9006 clinical trials and
sample collection were approved by the individual Institutional Review
Boards of POG member institutions and informed consent was obtained
from patients and/or their parents. Patients were enrolled
between January 1991 and November 1994. Cutoff for analysis was October
1996.
Molecular analyses.
Mononuclear cells were isolated from PB/BM samples by Ficoll-Hypaque
density centrifugation, lysed in 0.5 to 1.0 mL of Solution D22 and frozen at  70°C as cell
lysates. Frozen lysates were later transferred to the laboratory of one
of the authors (S.P.H.) at the University of Colorado Health Sciences
Center (UCHSC). After being stored at 70°C for 1 to 5 years,
the solution D lysates were thawed rapidly and total RNA was
extracted by the guanidinium acid lysis technique as previously
described,5,22 dissolved in diethylpyrocarbonate-treated
double-distilled H2O, and frozen at 70°C for
later analysis.
Two micrograms of total RNA was first treated with DNAse I (GIBCO-BRL,
Gaithersburg, MD) to eliminate any DNA present in the samples, and then
reverse transcribed to cDNA using random hexamer primers (Boehringer
Mannheim, Indianapolis, IN).23 One-half of the cDNA was
subjected to a two step nested PCR assay using E2A and
PBX1 primers flanking the site of mRNA fusion exactly as
described previously (Fig
1A).23 To insure that amplifiable RNA had been isolated,
the remaining one-half of the cDNA was used to amplify a portion of the
ubiquitously expressed ABL mRNA using primers and conditions
described previously.5,23 After amplification, one-fifth
(10 µL) of the second round E2A-PBX1 PCR product and the
ABL PCR product were size-fractionated by electrophoresis in
3% agarose gel. In a series of dilution experiments, the nested
E2A-PBX1 PCR assay consistently detected a
104 dilution of t(1;19)+ RCH-ACV RNA
into t(1;19) REH RNA, detected a
105 dilution in 50% to 70% of experiments and
never detected a 106 dilution. Hybridization of
immobilized nested PCR products with a radiolabeled internal
oligonucleotide did not increase the sensitivity or consistency
of detection; thus we relied on visualization of bands on ethidium
bromide-stained gels.
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| Fig 1.
Sensitivity of E2A-PBX1 PCR assay. The
E2A-PBX1 fusion mRNA and the relative location of amplification
primers (arrows) and detection oligonucleotides (lines) are
schematically depicted at the top. Below this are displayed a
photograph of an ethidium bromide-stained agarose gel of nested PCR
products and a Southern blot of this gel hybridized with radio-labeled
Probe-fus oligonucleotide (identical results were seen with
Probe-PBX1). Molecular size (MW) markers are indicated at left in base
pairs. Samples include t(1;19)+ RCH-ACV and
t(1;19) REH pre-B ALL cell lines, serial 10-fold
dilutions (101 to 106) of RCH-ACV RNA
into REH RNA, and reverse transcriptase (RT) and PCR negative (NEG)
controls.
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Numerous precautions were taken to decrease the chance of false
positive results. All RNAs were isolated before performing any
E2A-PBX1 PCR analyses at UCHSC. To further exclude false
positives, RNA was isolated in batches of 10 to 20 samples, which
included 1 to 2 samples from patients with t(9;22)+ ALL
treated on POG 9005/9006 who were involved in a parallel study of MRD
and from whom PB/BM samples had been collected contemporaneously and
handled identically in the same central laboratory at Stanford University. All PCR reactions were set up and run in a separate laboratory 2 miles away from that which had been used to isolate the
RNAs. After amplification, samples were transported back to the UCHSC
campus and PCR products were run on agarose gels and analyzed as
described above. Because of these precautions, the initial sample
handling, RNA isolation, PCR set up and amplification and gel analyses
were completely physically isolated from one another. If any
contamination was introduced during sample processing and/or
RNA isolation, one would anticipate that false positive results would
be seen in the t(9;22)+ samples.
Each run of PCR reactions included the following controls: RNAs from
RCH-ACV and REH pre-B ALL cell lines, a 104 dilution
of RCH-ACV RNA in REH RNA, 1 or 2 RNAs from t(9;22)+ ALL
patients treated on 9005/9006, a reverse transcription (RT) negative
control in which a sample containing water rather than input RNA was
carried through all steps beginning with the RT step, and a PCR
negative control in which water rather than input cDNA was subjected to
amplification. To consider a result valid, each of the following
requirements had to be met: a readily visible ABL PCR product
had to be present in the control PCR reaction, readily visible
E2A-PBX1-specific PCR products of the appropriate size had to
be amplified from RCH-ACV and the 104 dilution of
RCH-ACV, and each of the negative controls had to be negative. To
further validate positive samples, we required that each positive
sample be independently confirmed. Specifically, whenever available,
matched PB and BM samples from weeks 25 and/or 31 from an
individual patient were analyzed at the same time. If more than one
sample was available from a patient and all were concordant (all
positive or all negative), results were considered valid. If there was
any discordance, all samples were repeated 1 to 2 times. We required
two positive results to consider a sample positive. All PCR reactions
were performed without knowledge of treatment outcome and PCR results
were not available to the treating physicians.
Statistical methods.
To assess the prognostic significance of week 25/31 PCR results, we
compared the event-free survival (EFS) two ways. The first compared
patients with negative PCR results on all available assays (never
positive) against those with positive or mixed results (positive at
some times or sample sources but not at others). The second compared
patients who were positive on all available assays (never negative)
against those with negative or mixed results. By "never" we refer
to weeks 25 and 31 only. EFS was the dependent variable and the logrank
test24 was used. EFS curves were constructed by the method
of Kaplan-Meier.25 The only other formal statistical comparison was to assess the concordance of week 25 and week 31 PCR
results, using the exact conditional chi-square.26 It is important to realize that one cannot preclude the possibility of
selection bias in terms of the prognostic significance of PCR results,
because of the relatively large number of patients with missing data.
Further, given the small number of treatment failures, the power to
detect moderate-sized differences is relatively low.
| |
RESULTS |
Seventy-four patients with t(1;19)+ ALL were enrolled on
POG 9005 or 9006, 71 of whom were potentially available for analysis (1 was taken off study at week 4, 1 was lost to follow-up, and 1 relapsed
before week 31). Samples were received at week 25 and/or 31 from 68% of these patients (Table 1). By
study, samples collected at week 25 and/or 31 were received
from 17 of 30 t(1;19)+ patients enrolled in POG 9005 and 31 of 44 enrolled in POG 9006. In total, samples from 54% of
potential time points were available for analysis and matched PB and BM
analyses were available for 81% of these times. The outcome of
patients from whom samples were received is similar to that of all
t(1;19)+ patients enrolled in POG 9005/9006 (data not
shown).
A nested PCR assay that reproducibly detected a 104
to 105 dilution of t(1;19)+ into
t(1;19) RNA in pilot experiments was employed for
our studies (Fig 1). A 104 dilution of
t(1;19)+ RNA was included in each individual PCR
experiment. The PCR assay consistently detected a standard level of MRD
because we did not observe experiments in which the undiluted sample
was positive and the 104 dilution was negative.
E2A-PBX1 chimeric transcripts were detected in 41 of 148 (28%)
week 25 or 31 samples from t(1;19)+ ALL patients, 104 were
PCR and 3 were uninformative because an ABL
band was not amplified in the control PCR reaction (representative
results are shown in Fig 2). Nineteen of 20 samples from t(9;22)+ ALL patients analyzed were negative
for E2A-PBX1 chimeric transcripts and one was uninformative
because a control ABL band was not amplified. False positives
were not observed in the negative control cell line (REH), RT or PCR
negative controls included in each PCR run. There were no major
differences in PCR results in terms of clinical parameters including
patient age, presenting white blood count or NCI consensus risk status
(Table 2). Cryopreservation for up to 5 years did not materially affect the integrity of RNA isolated because a
correct-sized ABL PCR product was amplified from 179 of
183 (98%) frozen samples analyzed. There was no correlation between
length of sample storage and incidence of PCR positivity (data not
shown).
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| Fig 2.
Representative results of E2A-PBX1 PCR
assays. Photograph of an ethidium bromide-stained agarose gel
containing size-fractionated products of nested PCR for
E2A-PBX1 fusion mRNA. Molecular size (MW) markers are indicated at
left in base pairs. The sample identity is given at the top of the gel.
Samples include bone marrow (B) and/or peripheral blood (P)
from weeks 25 and/or 31 for patients 1 to 6, t(1;19)+ RCH-ACV and t(1;19) REH pre-B ALL
cell lines, a 104 dilution of RCH-ACV RNA into REH RNA,
and RT and PCR negative controls. The migration of the E2A-PBX1
PCR product is indicated at the right of the gel. For patient 2, two
independently isolated BM samples [Ba and Bb] were analyzed.
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We compared PCR results for the 65 pairs of PB and BM that were
collected at the same time point. Results were concordant for 51 pairs
(10 PB+/BM+, 41 PB/BM) and discordant for 14 (5 PB+/BM, 9 PB/BM+). The discordant sample pairs
were reproducibly discordant in all cases. Informative PCR results were
available from both weeks 25 and 31 for 32 of the patients. There was
no apparent association of PCR status at weeks 25 and 31 (Table 3). Approximately one-third of
patients who were PCR at week 25 were
PCR+ at week 31, quite similar to the percentage who were
PCR+ at week 25 and again positive at week 31 (P
value for the exact conditional chi-square test of association is > .99).
We analyzed EFS to determine if the presence or absence of amplifiable
E2A-PBX1 chimeric transcripts was predictive of treatment outcome. Because of the variability of PCR results, we compared several
different groupings to determine if any predictive value was apparent.
These included comparisons of patients for whom all available PCR
results were negative (n = 20) versus those in whom all results were
positive (n = 13) versus those with both negative and positive results
(n = 15); those who were PCR+ at week 25 and
PCR at week 31 (n = 6) versus those who were
PCR at week 25 and PCR+ at week 31 (n = 9); those who were PCR+ on all available assays (n = 13)
versus those who were always PCR plus those with
some positive and some negative results (n = 35); and those who were
PCR on all available assays (n = 20) versus those
who were always PCR+ plus those with some positive and some
negative results (n = 28). There were no significant differences in
treatment outcome between any of these comparisons. Selected EFS curves
are shown in Fig 3.
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| Fig 3.
EFS on POG 9005/9006 by t(1;19) PCR. (A) EFS of patients
for whom all available PCR results were positive versus that of
patients for whom all available results were negative plus those with
some positive and some negative PCR results (negative + mixed). (B) EFS of patients for whom all available PCR results were negative versus
that of patients for whom all available results were positive plus
those with some positive and some negative PCR results (positive + mixed). The data were analyzed by the Kaplan-Meier method and the
difference compared by the logrank test, the results of which are
included in tabular form within each graph.
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| |
DISCUSSION |
PCR analysis of MRD by amplification of E2A-PBX1 chimeric
transcripts at the end of consolidation therapy was not predictive of
treatment outcome for children with t(1;19)+ ALL treated on
POG 9005/9006. The lack of concordance between samples obtained
simultaneously from PB and BM and between samples obtained 6 weeks
apart (weeks 25 and 31) was surprising. These discrepancies could
potentially be due to the presence of levels of MRD at or near the
levels of detection of the assay (104 to
105). Given the elaborate precautions employed in
this study and inclusion of multiple negative controls, it is extremely
unlikely that our results are confounded by false positives due to PCR contamination. It is difficult to completely exclude false negative PCR
results, but we emphasize that all PCR assays included a
10-4 dilution of positive control RNA which consistently
gave positive results. Discrepant samples were reproducibly discrepant
in independently-performed PCR reactions. Thus, it seems unlikely that
the PCR assay employed in this study lacked sensitivity or
reproducibility. Most importantly, the lack of correlation between
clinical outcome and PCR results was quite consistent across all
comparisons. Based on these factors, we conclude that our results are
truly representative and not due to technical problems.
There were 6 time points for which a BM sample was
PCR, but a matched PB sample was not available.
Based on the discordance observed in some cases between simultaneously
obtained BM and PB samples, we considered the possibility that some or
all of these six data points might have been false negatives and
repeated the analyses with the results from these six data points
excluded. This did not alter our conclusions because there were no
significant differences in EFS based on PCR status in these analyses.
It is possible that the strategy we used to detect MRD was suboptimal
and that other strategies might be more predictive of outcome. We
decided to analyze samples obtained at the end of consolidation because
we wanted to assess disease burden before most relapses were likely to
occur (and only 1 of 74 patients relapsed before week 31) to avoid
missing identification of high-risk patients and the later opportunity
for early therapeutic intervention. As the intensive phase of treatment
had been completed by week 25, we hypothesized that the level of
leukemic cells would have decreased substantially by this time and that
levels of MRD might separate patients into cohorts at high and low risk
of subsequent relapse. This assumption may be false, or the decrease
may evolve over a longer period of time after completion of
consolidation and samples obtained later in therapy might have been
more informative. In this regard, it is important to emphasize that one
group found that Ig PCR assays performed at the end of
induction had clinical predictive value, but the same assays performed
at the time therapy was discontinued did not.27,28
Another potential problem is that we used a qualitative (positive
v negative) rather than a quantitative PCR assay. Recently several semiquantitative PCR assays for MRD detection have been developed. One strategy used for chimeric mRNAs such as BCR-ABL and AML1-ETO involves coamplification of known amounts of
artificial fusion templates of unique size and has been shown in
several studies to be more predictive of outcome than qualitative
PCR.29-32 Others have used recombinant phage plaque
counting techniques27,28 or limiting dilution assays with
multiple replicates33 to quantify MRD assessed by
amplifying unique Ig rearrangements. It is possible that such
approaches might better identify high-risk patients based on the level
of MRD or trends in the this level over time, rather than simply its
absence or presence. Finally, it is important to note that the overall
outcome for t(1;19)+ patients treated on POG 9005/9006 is
improved from prior POG studies and this high success rate combined
with limited sample numbers means that our study had a relatively low
power to detect differences in outcome. One problem encountered in our
study, as in similar longitudinal studies reported by others, is the incompleteness of sample collection at each time point. It is clear
that future studies of MRD, particularly those performed in large
cooperative groups, must develop methods to insure near complete sample
collection. If missing at random assumptions are not satisfied,
the validity of the method of choice, the Cox model,34 with
time dependent covariates, becomes severely biased without complete
data at every time point for nearly every patient.
Several small studies have described the use of E2A-PBX1 PCR to
assess MRD in t(1;19)+ ALL patients. Izraeli et
al16 analyzed 7 patients at one or two time points 6 to 36 months after attaining complete remission (CR). They found no obvious
correlation between PCR results and clinical outcome and 2 patients
remained in CR for >1 year after positive PCR assays at 24 and 29 months. There also was no obvious correlation of PCR results with
clinical outcome among 9 patients studied by Devaraj et
al14 with 2 patients remaining in CR for 20 to 32 months
after being PCR+ 5 to 6 months into therapy.14
Privitera et al17 assessed MRD in six t(1;19)+
patients. Five became PCR 3 to 6 months into therapy
and remained in CR at last follow-up (15 to 60 months after diagnosis).
One was persistently PCR+ during therapy, remained positive
when treatment was discontinued but continued in CR 5 years later.
Lanza et al18 assessed MRD in 3 t(1;19)+
patients; two became PCR early in therapy, while one
remained PCR+ 3 years after diagnosis.18
Despite this difference, all three patients remained in clinical and
hematological complete remission. Thus, none of the data available at
this time suggest that detection of MRD by E2A-PBX1 PCR in
t(1;19)+ ALL provides information that is sufficiently
predictive of treatment outcome to warrant alterations in patient
management.
Our study emphasizes several important facts that have relevance to MRD
analysis. First, discordant PCR results were observed at 22% of the
time points for which results were available from simultaneously
obtained PB and BM specimens (9 PB /BM+
and 5 PB+/BM ), suggesting that
conclusions based upon PCR results obtained from PB only or BM only are
problematic. Although most studies of MRD in acute leukemia have
focused on BM samples, two other studies analyzed MRD detection in
matched BM/PB samples from patients with t(9;22)+ ALL.
Similar to our findings, Radich et al35 reported concordant PCR results from 23/31 patients, while 5 were
BM+/PB and 3 BM/PB+.35 In contrast, van
Rhee et al36 found that while that the number of
BCR-ABL transcripts in paired PB and BM samples did not differ
significantly overall, four patients were
PB/BM+, while none were
PB+/BM.36
Second and most important, our results underscore the danger in
extrapolating results from one method of PCR detection of MRD to
another. In several settings, experimental evidence suggests that PCR
detection of MRD by amplification of chimeric mRNAs produced by
chromosomal translocations provides clinically useful information which
may have therapeutic implications. Detection of MRD by amplification of
BCR-ABL fusion mRNAs in patients with CML after BMT has been performed in numerous studies and a consensus is emerging that patients
who are reproducibly PCR+ after the early post-BMT phase
are at increased risk of relapse when compared to those who are
reproducibly PCR.37,38 Similarly,
detection of MRD by amplification of PML-RARA fusion
transcripts at the end of therapy appears to provide useful clinical
information in patients with acute promyelocytic
leukemia.39-44 On the other hand, the presence of
amplifiable AML1-ETO fusion transcripts in most or all
t(8;21)+ AML patients long after completion of chemotherapy
or BMT indicates that qualitative PCR results are not predictive of
treatment outcome in this subset of AML, although a recent small
series provides preliminary data suggesting that quantitative PCR may
be more informative.32,45-48 Thus, it is clear that the
potential clinical utility of each individual PCR MRD assay needs to be
rigorously determined before results are used to modify treatment of
individual patients or groups of patients.
| |
FOOTNOTES |
Submitted June 20, 1997;
accepted September 29, 1997.
Supported by grants from The Southern California Children's Cancer
Service and Couples Against Leukemia, Los Angeles, CA (S.P.H.), The
Maxfield Foundation, Saratoga, CA (S.P.H.), and the Ladies Auxiliary
to the Veterans of Foreign Wars, Denver, CO (S.P.H.). S.P.H. was
supported in part by a Physicians Research Training Fellowship from the
American Cancer Society and a National Research Service Award from the
National Institutes of Health (Bethesda, MD). S.P.H. is the recipient
of a BLOOD/ASH Scholar Award. B.M.C. is the Rebecca Jean Slye Professor
of Pediatric Oncology and is supported in part by the Midwest Athletes
Against Childhood Cancer (MACC) Fund. The National Cancer
Institue grant numbers for participating authors:
CA-09184, CA-33603, CA-32053, CA-25408, CA-20549, CA-03161, CA-15989,
CA-29139, CA-30969, CA-37379, CA-49605. For complete grant information
see the Appendix.
Address reprint requests to Stephen P. Hunger, MD (9000/9005/9006), c/o
POG Operations Office, 645 N Michigan Ave, Suite 910, Chicago, IL
60610.
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.
| |
ACKNOWLEDGMENT |
We gratefully acknowledge the invaluable assistance of the physicians
who treated these patients and, most importantly, the patients and
their parents, without whom these studies would not have been possible.
We also thank Sharon Murphy for her encouragement and interest in this
project. We are particularly indebted to Qi Wei for technical
assistance and the use of his
laboratory.
| |
REFERENCES |
1.
Campana D,
Pui C-H:
Detection of minimal residual disease in acute leukemia: Methodologic advances and clinical significance.
Blood
85:1416,
1995[Free Full Text]
2.
Hunger SP:
Chromosomal translocations involving the E2A gene in acute lymphoblastic leukemia: Clinical features and molecular pathogenesis.
Blood
87:1211,
1996[Free Full Text]
3.
Kamps MP,
Murre CM,
Sun X-H,
Baltimore D:
A new homeobox gene contributes the DNA binding domain of the t(1;19) translocation protein in pre-B ALL.
Cell
60:547,
1990[Medline]
[Order article via Infotrieve]
4.
Nourse J,
Mellentin JD,
Galili N,
Wilkinson J,
Stanbridge E,
Smith SD,
Cleary ML:
Chromosomal translocation t(1;19) results in synthesis of a homeobox fusion mRNA that codes for a potential chimeric transcription factor.
Cell
60:535,
1990[Medline]
[Order article via Infotrieve]
5.
Hunger SP,
Galili N,
Carroll AJ,
Crist WM,
Link M,
Cleary ML:
The t(1;19)(q23;p13) results in consistent fusion of E2A and PBX1 coding sequences in acute lymphoblastic leukemias.
Blood
77:687,
1991[Abstract/Free Full Text]
6.
Pui C-H,
Raimondi SC,
Hancock ML,
Rivera GK,
Ribeiro RC,
Mahmoud HH,
Sandlund JT,
Crist WM,
Behm FG:
Immunologic, cytogenetic, and clinical characterization of childhood acute lymphoblastic leukemia with the t(1;19)(q23; p13) or its derivative.
J Clin Oncol
12:2601,
1994[Abstract/Free Full Text]
7.
Crist W,
Boyett J,
Jackson J,
Vietti T,
Borowitz M,
Chauvenet A,
Winick N,
Ragab A,
Mahoney D,
Head D,
Iyer R,
Wagner H,
Pullen J:
Prognostic importance of the pre-B immunophenotype and other presenting features in B-lineage childhood acute lymphoblastic leukemia: A Pediatric Oncology Group study.
Blood
74:1252,
1989[Abstract/Free Full Text]
8.
Crist WM,
Carroll AJ,
Shuster JJ,
Behm FG,
Whitehead M,
Vieti TJ,
Look AT,
Mahoney D,
Ragab A,
Pullen DJ,
Land VJ:
Poor prognosis of children with Pre-B acute lymphoblastic leukemia is associated with the t(1;19)(q23;p13). A Pediatric Oncology Group Study.
Blood
76:117,
1990[Abstract/Free Full Text]
9. Raimondi SC, Behm FG, Roberson PK, Williams DL Pui C-H, Crist WM,
Look AT, Rivera GK: Cytogenetics of pre-B-cell acute lymphoblastic
leukemia with emphasis on prognostic implications of the t(1;19). J
Clin Oncol 8:1380, 1990
10.
Mellentin JD,
Nourse J,
Hunger SP,
Smith SD,
Cleary ML:
Molecular analysis of the t(1;19) breakpoint cluster region in pre-B cell ALL.
Genes Chromosomes Cancer
2:239,
1990[Medline]
[Order article via Infotrieve]
11.
Numata S,
Kato K,
Horibe K:
New E2A/PBX1 fusion transcript in a patient with t(1;19)(q23;p13) acute lymphoblastic leukemia.
Leukemia
7:1441,
1993[Medline]
[Order article via Infotrieve]
12.
Izraeli S,
Henn T,
Strobl H,
Ludwig WD,
Kovar H,
Haas OD,
Harbott J,
Bartram CR,
Gardner H,
Lion T:
Expression of identical E2A-PBX1 fusion transcripts occurs in both pre-B and early pre-B immunological subtypes of childhood acute lymphoblastic leukemia.
Leukemia
7:2054,
1993[Medline]
[Order article via Infotrieve]
13.
Privitera E,
Kamps MP,
Hayashi Y,
Inaba T,
Shapiro LH,
Raimondi SC,
Behm F,
Hendershot L,
Carroll AJ,
Baltimore D,
Look AT:
Different molecular consequences of the 1;19 chromosomal translocation in childhood B-cell precursor acute lymphoblastic leukemia.
Blood
79:1781,
1992[Abstract/Free Full Text]
14.
Devaraj PE,
Foroni L,
Janossy G,
Hoffbrand AV,
Secker-Walker LM:
Expression of the E2A-PBX1 fusion transcripts in t(1;19)(q23;p13) and der(19)t(1;19) at diagnosis and in remission of acute lymphoblastic leukemia with different B lineage immunophenotypes.
Leukemia
9:821,
1995[Medline]
[Order article via Infotrieve]
15.
Troussard X,
Rimokh R,
Valensi F,
Leboeuf D,
Fenneteau O,
Guitard A-M,
Manel A-M,
Schillenger F,
Leglise C,
Brizard A,
Gardais J,
Lessard M,
Flandrin G,
MacIntyre E:
Heterogeneity of t(1;19)(q23;p13) acute leukemias.
Br J Haematol
89:516,
1995[Medline]
[Order article via Infotrieve]
16.
Izraeli S,
Janssen WSG,
Haas OA,
Harbott J,
Brok-Simoni F,
Walher JU,
Kovar H,
Henn T,
Ludwig W-D,
Reiter A,
Rechavi G,
Bartram CR,
Gadner H,
Lion T:
Detection and clinical relevance of genetic abnormalities in pediatric acute lymphoblastic leukemia: A comparison between cytogenetic and polymerase chain reaction analyses.
Leukemia
7:671,
1993[Medline]
[Order article via Infotrieve]
17.
Privitera E,
Rivolta A,
Ronchetti D,
Mosna G,
Giudidi G,
Biondi A:
Reverse transcriptase/polymerase chain reaction follow-up and minimal residual disease detection in t(1;19)-positive acute lymphoblastic leukemia.
Br J Haematol
92:653,
1996[Medline]
[Order article via Infotrieve]
18.
Lanza C,
Gottardi E,
Gaidano G,
Vivenza C,
Parziale A,
Perfetto F,
Fornaci M,
Barisone E,
Madon G,
Saglio G:
Persistence of E2A/PBX1 transcripts in t(1;19) childhood acute lymphoblastic leukemia: Correlation with chemotherapy intensity and clinical outcome.
Leuk Res
20:441,
1996[Medline]
[Order article via Infotrieve]
19.
Kobayashi H,
Maseki N,
Homma C,
Sakurai M,
Kaneko Y:
Clinical significance of chromosome abnormalities in childhood acute lymphoblastic leukemia in Japan.
Leukemia
8:1954,
1994
20.
Lampert F,
Harbott J,
Ritterbach J,
Schellong G,
Ritter J,
Creutzig U,
Riehm H,
Rieter A:
Karyotypes in acute childhood leukemias may lose prognostic significance with more intensive and specific chemotherapy.
Cancer Genet Cytogenet
54:277,
1991[Medline]
[Order article via Infotrieve]
21.
Camitta B,
Mahoney D,
Leventhal B,
Lauer SJ,
Shuster JJ,
Adair S,
Civin C,
Munoz L,
Steuber P,
Strother D:
Intensive intravenous methotrexate and mercaptopurine treatment of higher-risk non-T, non-B acute lymphocytic leukemia: A Pediatric Oncology Group study.
J Clin Oncol
12:1383,
1994[Abstract]
22.
Chomczynski P,
Sacchi N:
Single-step method of RNA isolation by acid guanidinium-thiocyanate-phenol-chloroform extraction.
Anal Biochem
162:156,
1987[Medline]
[Order article via Infotrieve]
23. Hunger SP, Sun T, Boswell AF, Carroll AJ, McGavran L:
Hyperdiploidy and E2A-PBX1 fusion in an adult with
t(1;19)+ acute lymphoblastic leukemia: Case report and
review of the literature. Genes Chromosomes Cancer (in
press)
24.
Peto R,
Pike MC,
Armitage P,
Breslow NE,
Cox DR,
Howard SV,
Mantel N,
McPherson K,
Peto J,
Smith PG:
Design and analysis of randomized clinical trials requiring prolonged observation of each patient. II. Analysis and examples.
Br J Cancer
35:1,
1977[Medline]
[Order article via Infotrieve]
25.
Kaplan EL,
Meier P:
Nonparametric estimation from incomplete observations.
J Am Stat Assoc
53:457,
1958
26. Lehmann EL: Nonparametrics: Statistical Methods Based on Ranks.
San Francisco, CA, Holin-Day, 1975
27.
Yamada M,
Wasserman R,
Lange B,
Reichard BA,
Womer RB,
Rovera G:
Minimal residual disease in childhood B-lineage lymphoblastic leukemia.
N Engl J Med
323:448,
1990[Abstract]
28.
Ito Y,
Wasserman R,
Galili N,
Reichard BA,
Shane S,
Lange B,
Rovera G:
Molecular residual disease status at the end of chemotherapy fails to predict subsequent relapse in children with B-lineage acute lymphoblastic leukemia.
J Clin Oncol
11:546,
1993[Abstract/Free Full Text]
29.
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
82:1929,
1993[Abstract/Free Full Text]
30.
Malinge MC,
Mahon FX,
Delfau MH,
Daheron L,
Kitzis A,
Guilhot F,
Tanzer J,
Grandchamp B:
Quantitative determination of the hybrid Bcr-Abl RNA in patients with chronic myelogenous leukaemia under interferon therapy.
Br J Haematol
82:701,
1992[Medline]
[Order article via Infotrieve]
31.
Thompson JD,
Brodsky I,
Yunis JJ:
Molecular quantification of residual disease in chronic myelogenous leukemia after bone marrow transplantation.
Blood
79:1629,
1992[Abstract/Free Full Text]
32.
Tobal K,
Yin JAL:
Monitoring of minimal residual disease by quantitative reverse transcriptase-polymerase chain reaction for AML1-MTG8 transcripts in AML-M2 with t(8;21).
Blood
88:3704,
1996[Abstract/Free Full Text]
33.
Roberts WM,
Estrov Z,
Ouspenskaia MV,
Johnston DA,
McClain KL,
Zipf TF:
Measurement of residual leukemia during remission in childhood acute lymphoblastic leukemia.
N Engl J Med
226:317,
1997
34. Cox DR: Regression models and life-tables. JR Stat Soc B34:187,
1972
35.
Radich J,
Gehly G,
Lee A,
Avery R,
Bryant E,
Edmands S,
Gooley T,
Kessler P,
Kirk J,
Ladne P,
Thomas ED,
Appelbaum FR:
Detection of bcr-abl transcripts in Philadelphia chromosome-positive acute lymphoblastic leukemia after marrow transplantation.
Blood
89:2602,
1997[Abstract/Free Full Text]
36.
van Rhee F,
Marks DI,
Lin F,
Szydlo RM,
Hochhaus A,
Treleaven J,
Delord C,
Cross NCP,
Goldman JM:
Quantification of residual disease in Philadelphia-positive acute lymphoblastic leukemia: Comparison of blood and bone marrow.
Leukemia
9:329,
1995[Medline]
[Order article via Infotrieve]
37.
Lion T:
Clinical implications of qualitative and quantitative polymerase chain reaction analysis in the monitoring of patients with chronic myelogenous leukemia.
Bone Marrow Transplant
14:505,
1994[Medline]
[Order article via Infotrieve]
38.
Radich JP,
Gehly G,
Gooley T,
Bryant E,
Clift RA,
Collins S,
Edmands S,
Kirk J,
Lee A,
Kessler P,
Schoch G,
Buckner CD,
Sullivan KM,
Appelbaum FR,
Thomas ED:
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
85:2632,
1995[Abstract/Free Full Text]
39.
Lo Coco F,
Diverio D,
Pandolfi PP,
Biondi A,
Rossi V,
Avvisati G,
Rambaldi A,
Arcese W,
Petti MC,
Meloni G,
Mendelli F,
Grignani F,
Masera G,
Barbui T,
Pilicci PG:
Molecular evaluation of residual disease as a predictor of relapse in acute promyelocytic leukemia.
Lancet
340:1437,
1992[Medline]
[Order article via Infotrieve]
40.
Huang W,
Sun GL,
Li XS,
Cao Q,
Lu Y,
Jang GS,
Zhang FQ,
Chai JR,
Wang ZY,
Waxman S,
Chen Z,
Chen S-J:
Acute promyelocytic leukemia: Clinical relevance of two major PML-RAR alpha isoforms and detection of minimal residual disease by retrotranscriptase/polymerase chain reaction to predict relapse.
Blood
82:1264,
1993[Abstract/Free Full Text]
41.
Miller WH Jr,
Levine K,
DeBlasio A,
Frankel SR,
Dmitrovsky E,
Warrell RP Jr:
Detection of minimal residual disease in acute promyelocytic leukemia by a reverse transcription polymerase chain reaction assay for the PML/RAR-alpha fusion mRNA.
Blood
82:1689,
1993[Abstract/Free Full Text]
42.
Laczika K,
Mitterbauer G,
Korninger L,
Knobl P,
Schwarzinger I,
Kapiotis S,
Haas OA,
Kyrle PA,
Pont J,
Oehler L,
Purtscher B,
Thalhammer F,
Lechner K,
Jaeger U:
Rapid achievement of PML-RAR alpha polymerase chain reaction (PCR)-negativity by combined treatment with all-trans-retinoic acid and chemotherapy in acute promyelocytic leukemia: A pilot study.
Leukemia
8:1,
1994[Medline]
[Order article via Infotrieve]
43.
Diverio D,
Pandolfi PP,
Biondi A,
Avvisati G,
Petti MC,
Mandelli F,
Pelicci G,
Lo Coco F:
Absence of reverse transcription-polymerase chain reaction detectable residual disease in patients with acute promyelocytic leukemia in long-term remission.
Blood
82:3556,
1993[Abstract/Free Full Text]
44. Fukitani H, Naoe T, Yoshida H, Kiyoi H, Miyawaki S, Morishita H,
Sano F, Kamibayashi H, Matsue K, Miyake T, Hasegawa S, Ueda Y, Kato Y,
Kabayashi H, Shimazaki M, Kurane R, Sakota H, Masaki K, Wakayama T,
Tohyama K, Nonaka Y, Natori and the Leukemia Study Group of the
Ministry of Health and Welfare (Kouseisho): Prognostic significance of
the RT-PCR assay of PML-RARA transcripts in acute promyelocytic
leukemia. Leukemia 9: 588, 1995
45.
Chang KS,
Fan YH,
Stass SA,
Estey EH,
Wang G,
Trujillo JM,
Erickson P,
Drabkin H:
Expression of AML1-ETO fusion transcripts and detection of minimal residual disease in t(8;21)-positive acute myeloid leukemia.
Oncogene
8:983,
1993[Medline]
[Order article via Infotrieve]
46.
Nucifora G,
Larson RA,
Rowley JD:
Persistence of the 8;21 translocation in patients with acute myeloid leukemia type M2 in long-term remission.
Blood
82:712,
1993[Abstract/Free Full Text]
47. Maruyama F, Stass SA, Estey EH, Cork A, Hirano M, Ino T,
Freireich, EJ, Yang P, Chang KS: Detection of AML1/ETO fusion transcript as a tool for diagnosing t(8;21) positive acute myelogenous leukemia. Leukemia 8:40, 1994
48.
Kusec R,
Laczika K,
Knobl P,
Friedl J,
Greinix H,
Kahls P,
Linkesch W,
Schwarzinger I,
Mitterbauer G,
Purtscher B,
Haas OA,
Lechner K,
Jaeger U:
AML1/ETO fusion mRNA can be detected in remission blood samples of all patients with t(8;21) acute myeloid leukemia after chemotherapy or autologous bone marrow transplantation.
Leukemia
8:735,
1994[Medline]
[Order article via Infotrieve]
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Abstract
Introduction
Abstract