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Prepublished online as a Blood First Edition Paper on December 5, 2002; DOI 10.1182/blood-2002-05-1357.
 Previous Article | Table of Contents | Next Article 
Blood, 1 April 2003, Vol. 101, No. 7, pp. 2521-2528
CLINICAL OBSERVATIONS, INTERVENTIONS, AND THERAPEUTIC TRIALS
Quantitative real-time RT-PCR analysis of PML-RAR mRNA in
acute promyelocytic leukemia: assessment of prognostic significance in
adult patients from intergroup protocol 0129
Robert E. Gallagher,
Beow Y. Yeap,
Wanli Bi,
Kenneth J. Livak,
Nike Beaubier,
Sreenivas Rao,
Clara D. Bloomfield,
Frederick R. Appelbaum,
Martin S. Tallman,
James L. Slack, and
Cheryl L. Willman
From the Departments of Oncology and Medicine,
Montefiore Medical Center and Albert Einstein Cancer Center, Bronx, NY;
the Department of Medicine, Massachusetts General Hospital and Harvard
Medical School, Boston, MA; Applied Biosystems, Foster City, CA; The
Ohio State University Comprehensive Cancer Center, Columbus, OH; the
Fred Hutchinson Cancer Research Center, Seattle, WA; the Division of
Hematology/Oncology, Department of Medicine, Northwestern University
Medical School, Chicago, IL; the Department of Medicine, Roswell Park
Cancer Institute, Buffalo, NY; the Departments of Pathology and Cell
Biology and the University of New Mexico Cancer Center, University of
New Mexico, Albuquerque, NM.
 |
Abstract |
The potential prognostic value of quantitative real-time reverse
transcription-polymerase chain reaction (RT-PCR [qrtPCR]) measurements of PML-RAR mRNA in acute promyelocytic leukemia was
retrospectively assessed before treatment and at 3 posttreatment intervals in 123 patients on intergroup protocol 0129. The primary measure was the PML-RARGAPDH normalized quotient (NQ),
that is, PML-RAR mRNA copies divided by
glyceraldehyde-3'-phosphate dehydrogenase (GAPDH) mRNA copies.
Only samples with more than 2.5 × 105 copies of the
housekeeping gene GAPDH mRNA (detection sensitivity exceeding
104) were considered NQ evaluable. With RNA from
low-density selected cells, paired peripheral blood (PB) and bone
marrow samples (n = 140) had comparable NQs
(P < .001). Before treatment, high NQ was
associated with short-form PML-RAR
(P < .001), but not with white blood cell
count or clinical outcome. Following treatment, NQ was lower
in all-trans retinoic acid-induced complete remission (CR)
than chemotherapy-induced CR (P = .018) and at
first test after consolidation chemotherapy
(P = .037). After consolidation chemotherapy, patients
with NQ exceeding 10 5 had 4.1-fold increased relapse risk
(P = .008); however, 73% of patients who experienced
relapse had NQ lower than 105. In the follow-up
period (FUP), any NQ exceeding 105 and
106 had 17.5-fold and 7.6-fold increased relapse risk,
respectively (P < .001), while no
gradation of relapse risk (approximately 18%) could be identified at
NQ lower than 106, including NQ. These
results indicate that qrtPCR monitoring of PML-RAR NQ can identify
patients at high risk of relapse and suggest that clinically practical
PB NQ monitoring at more frequent FUP intervals may improve predictive
accuracy for relapse or continuing CR in patients with persistent,
fluctuating minimal residual disease levels.
(Blood. 2003;101:2521-2528)
© 2003 by The American Society of Hematology.
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Introduction |
Recent improvements in the treatment of acute
promyelocytic leukemia (APL), which accounts for about 10% of acute
myeloid leukemias, have increased the long-term remission and apparent cure rate to approximately 70%.1-6 The primary factor in
this improvement has been the addition of all-trans retinoic
acid (ATRA) to anthracycline-containing chemotherapy, with the best
results reported in clinical trials in which ATRA is given concurrently with chemotherapy during the remission-induction
phase.2,4-6 Additionally, 2 trials have provided
evidence that, after various types of consolidation therapy, ATRA
maintenance therapy has a positive effect in securing long-term
remissions.1,4 Nevertheless, disease relapse
has been a major contributor to treatment failure and death.
A powerful tool that has been used to attempt to prospectively identify
patients at high risk of experiencing relapse is reverse transcription-polymerase chain reaction (RT-PCR) monitoring of mRNA
encoding the APL-specific fusion protein PML-RAR in order to detect
minimal residual disease (MRD) (reviewed in Diverio et
al11). Early studies, using RT-PCR assays with a
sensitivity of 103 to 104, found that patients
who were persistently PCR+ after completing induction and
consolidation therapy with ATRA plus chemotherapy regimens had a high
incidence of relapse within a few months (approximately
75%).7-10 Conversely, following consolidation chemotherapy, patients who were PCR experienced relapse
much less frequently, with incidences of 17% and 27% in 2 large
clinical trial studies.5,11 However, because fewer than
10% of patients in these studies were PCR+ after
consolidation, the total number of relapses were greater in
the PCR patients. These considerations pointed out the
importance of continued PCR monitoring in the postconsolidation
follow-up period (FUP). In one of these studies, only 8 (6%) of 142 cases with 2 or more PCR tests during the FUP
subsequently experienced relapse, while 20 of 21 cases that converted
to PCR+ experienced relapse (median follow-up, 18 months).11 These results were recently confirmed in
another study with long-term follow-up (median, 63 months) in which
only 3 (7%) of 41 cases with 2 or more PCR tests
experienced relapse, while 4 of 4 cases with 2 or more PCR+
assays experienced relapse.12 The therapeutic relevance of these findings was further supported by a pilot study in which the
administration of additional treatment after detection of PML-RAR by
PCR produced superior clinical outcome compared with a historical
control group in which salvage therapy was administered only after
hematologic relapse.13 The concept of basing salvage therapy on the detection of persistent or recurrent MRD has been incorporated in the principal Italian protocol for de novo
APL,13 and the same criteria have been proposed for the
evaluation of novel therapeutic approaches with the goal of increasing
therapeutic index.14
In the current study, we used quantitative real-time RT-PCR (qrtPCR) to
study samples from intergroup protocol 0129 (INT0129), in which
patients were initially randomized to induction therapy with either
ATRA or chemotherapy (daunorubicin and cytarabine [DA]) and,
following 2 courses of DA consolidation therapy, were rerandomized to
either ATRA maintenance for 1 year or no further treatment (termed
"observation").1 In preclinical experiments using cells and RNA from the APL cell lines NB4 and UF-1 that express
the long (L) and short (S) forms of PML-RAR,
respectively,15,16 we established assay
conditions and evaluation criteria that enabled us to confidently
measure PML-RAR mRNA expression levels over a 6 log-linear range,
excluding from analysis all samples with a detection sensitivity of
less than 104.17 This methodology has the
potential of extending the effectiveness of MRD monitoring in APL to be
higher than previously applied, nonquantitative manual RT-PCR
(mrtPCR) procedures by providing a more precise estimate of relapse
risk from defined MRD levels detected, on average, with greater than
10-fold higher sensitivity. In addition to identifying a threshold for
high relapse risk, it might identify a low-level MRD threshold below
which the risk of relapse is minimal, as suggested by previous findings
of persistent low levels of PML-RAR transcripts in long-term
complete remission (CR) cases of APL using high-sensitivity
mrtPCR.18 Also, quantification of PML-RAR mRNA levels
prior to and following initial therapy might be of prognostic value.
The results of this investigation indicate that this methodology indeed
has the capacity to identify cases at defined high risk of relapse
following the completion of consolidation chemotherapy. They also
indicate, however, that other potential prognostic applications are
limited by an apparent lack of a tight relationship between PML-RAR
mRNA levels and clinical outcome. In some instances, it may be possible
to overcome these limitations by performing more frequent assays,
possibly abetted by our additional finding that qrtPCR assays using RNA extracted from peripheral blood cells are nearly as effective as those
from less conveniently obtained bone marrow.
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Patients, materials, and methods |
Patients and samples
Patient materials used for this study were obtained from 123 patients (aged 15 years or older) registered to protocol INT0129 by the
Eastern Cooperative Oncology Group (ECOG), the Cancer and Leukemia Group B (CALGB), and the Southwest Oncology Group
(SWOG) under a uniform consent form approved by the
institutional review boards of all participating institutions. This
patient subgroup was derived from a previously reported group of 203 patients determined to have either the S form or L form of PML-RAR,
a requirement for RT-PCR monitoring.19 Selection for the
study group was based solely on the availability of more than 1 postinduction treatment sample. Of the 123 pretreatment cases, 116 achieved CR and 111 completed consolidation therapy. Of the latter
group, 97 cases were randomized to either ATRA maintenance (47 cases;
24 and 23 cases treated with ATRA or DA induction, respectively) or
observation (50 cases; 30 and 20 cases treated with ATRA or DA
induction, respectively). Fourteen additional patients who received no
postconsolidation treatment owing either to assignment or to default
were monitored in the FUP and, for the current analyses, were combined
with the observation group (64 total no-treatment cases). Of these
cases, 9 had been induced with ATRA, 4 with DA, and 1 with ATRA
(failure) followed by DA induction therapy.
Collection of bone marrow (BM) and peripheral blood (PB)
samples was not mandated by INT0129. Nevertheless, 822 samples
from the 123-patient study group (median, 6 samples per patient; range, 2-27 samples) were retrospectively available from recommended collection intervals for RT-PCR analysis. The analyses in this report
used 593 samples from 4 strictly defined monitoring intervals: (1)
before any antileukemic therapy (pretreatment); (2) after achieving
clinical and hematological remission on either ATRA or DA induction
therapy but before the administration of consolidation chemotherapy (at
CR); (3) after completion of 2 courses of consolidation chemotherapy but before the second protocol randomization (after consolidation chemotherapy); and (4) during the follow-up period after
the second protocol randomization (FUP). The checkpoint used
after consolidation chemotherapy was the first evaluable sample after
completion of consolidation therapy. The FUP samples were
irregularly collected at recommended 6-month intervals for the first 2 years and at 12-month intervals for up to 5 years, including samples up
to the time of clinical relapse or to the last official entry in the
central protocol database of continuing CR (CCR).
Sample processing and RT-PCR procedures
Low-density mononuclear cells (LDMNCs) (density, 1.077 g/mL or lower) were prepared from BM and PB19 and were
either immediately extracted in guanidinium buffer or viably conserved.
Total RNA was isolated from fresh LDMNCs (5 to 20 × 106
cells) by a modification of the guanidine-HCl extraction/cesium chloride density-gradient procedure, by a modification of the acid phenol extraction method,19 or from thawed,
fresh-frozen LDMNCs by means of 1 of 2 proprietary column affinity
procedures (RNAqueous kits [Ambion, Austin, TX] or RNeasy
kits [Qiagen, Valencia, CA]). All RNAs prepared by initial
guanidinium extraction were repurified by means of an RNeasy kit
(Qiagen) to provide uniform material for cDNA synthesis
reactions.17 Reverse transcriptase reactions,
using antisense gene-specific primers for first-strand cDNA synthesis,
and PCR amplification were performed at a single site (by W.B.
at Applied Biosystems [ABI], Foster City, CA), as previously
described.17
The semiautomated collection of qrtPCR amplification data by TaqMan
technology, using the ABI PRISM 7700 DNA Sequence Detection System was
as previously reported.17,20 Briefly, PML-RAR and glyceraldehyde-3'-phosphate dehydrogenase (GAPDH) transcript copy numbers were determined by the first PCR cycle during real-time analysis in which the initiation of exponential cDNA template amplification exceeds background by 10-fold (the threshold cycle [CT]) and by reference to plasmid DNA standard curves, in
which a single template copy is detectable at a CT of
40.20 In this study, we considered as positive any sample
with an average CT for PML-RAR lower than 40, even if 1 or 2 of the triplicate values was 40. Although such low-level activity
could represent nonspecific background, we did not observe this in a
large series of negative controls.17 Thus, these low-level
values are likely to reflect the stochastic nature of PCR assays near
the limit of detection,17,21,22 and they were regarded as
quantifiable positives. Reported PML-RAR and GAPDH copy numbers were
the geometric average of triplicate (infrequently, duplicate)
determinations derived from independent PCRs performed from the same
cDNA. PML-RAR values are reported as the normalized quotient (NQ),
derived by dividing the PML-RAR copy number by the GAPDH copy
number. The use of GAPDH for intersample normalization was based on a
preclinical study in which we found GAPDH mRNA to be equally expressed,
with little variation in LDMNCs from normal BM and PB.17
Quantitative real-time RT-PCR sample assessment
In the preclinical study, we determined that a GAPDH copy number
of 2.5 × 105 corresponds to a detection sensitivity of 1 leukemic cell in 104 LDMNCs.17 In this report,
samples with fewer than 2.5 × 105 GAPDH copies,
reflecting low RNA quantity and/or poor efficiency of cDNA synthesis,
were considered nonevaluable and excluded from analysis. By this
criteria, 92 (16%) of 593 samples from the 4 monitoring intervals were
nonevaluable. Although a higher fraction of PB than BM samples were
nonevaluable (21% versus 13%; P = .009), the fractions
of PB and BM samples with GAPDH copy numbers in lower (greater than
2.5 × 105 to 2.5 × 106) and upper
(greater than 2.5 × 106) evaluable sensitivity ranges
were equal (P = .777). Thus, a total of 501 evaluable
samples, 328 BM, 171 PB, and 2 BM + PB were available for assessment at
the 4 specified monitoring intervals.
Statistical methods
The distribution of GAPDH ranges between BM and PB was compared
by Fisher exact test.23 Agreement between NQs from
simultaneous BM and PB samples and the association between NQs before
treatment and at CR were assessed by Spearman rank-order
correlation.24 Difference in the distribution of NQs
between 2 patient groups was evaluated by the Wilcoxon rank-sum
test.24 Disease-free survival (DFS) for the analysis after
consolidation therapy is defined as the time from achievement of CR per
protocol INT0129 to relapse, death, or last follow-up, while DFS for
the FUP analysis is from randomization for the maintenance phase or end
of consolidation therapy among the nonrandomized patients. Proportional
hazards regression of DFS was used to analyze the predictive value of NQ cutoff levels while adjusting for treatment, PML-RAR type, and
white blood cell (WBC) count at presentation; NQ during FUP was analyzed as a binary time-varying covariate according to cutoff level.23 DFS rates were estimated by the method of Kaplan
and Meier.25 All values for P are based on a
2-sided hypothesis owing to the exploratory nature of the statistical
tests and, thus, should not be interpreted by strict conventional criteria.
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Results |
Patient characteristics
Four important characteristics (age, sex distribution,
WBC count, and DFS follow-up) of the 123-patient study group were
similar to those of 344 total adult protocol patients (Table
1).1,26 The proportion of
study group patients who received ATRA as induction or maintenance
therapy deviated from the equal randomization in INT0129; however, the
impact of ATRA on DFS was adjusted by multivariate modeling. Also, the
fraction of patients in CCR was higher in the study group than in total
adult patients, 66 (54%) of 123 versus 129 (38%) of 344. Accordingly,
we also compared the distribution of characteristics of
patients in the 2 cohorts who achieved CR, but found no
differences. Finally, there was no difference in the
distribution of L- or S-form PML-RAR types from 203 previously reported adult INT0129 patients,19 further indicating the
representative nature of the study group.
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Table 1.
Characteristics of 123 patients monitored with serial
PML-RAR NQ determinations compared with 344 overall adult patients
on protocol INT0129
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Comparison of samples from bone marrow versus peripheral blood and
from 4 monitoring intervals
The comparable distribution of GAPDH copy numbers in PB and BM
between the lower and upper evaluable sensitivity ranges ("Patients, materials, and methods") suggested that PB might provide similar sensitivity to BM for detecting PML-RAR transcripts. To evaluate this further, we compared PML-RAR NQs for 140 points at which evaluable RNA samples were available from both BM and PB, and a strong
correlation of NQs between these simultaneous pairs was found (Spearman
rank-order coefficient = 0.84, P < .001) (Figure 1). Moreover, a good correlation was
found between NQs of 102 sample pairs at posttreatment time points with
MRD (Spearman = 0.67, P < .001). Overall, PB
was only slightly less effective than BM for monitoring PML-RAR NQs,
and on the basis of this assessment, we substituted NQ data from
evaluable PB samples for which a BM sample was not available or
not evaluable.
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| Figure 1.
Paired analysis of PML-RAR NQs from 140 simultaneously obtained, evaluable BM and PB samples from INT0129
patients.
Values obtained by dividing PML-RAR copy number by GAPDH
copy number for BM (abscissa) and PB (ordinate) are co-plotted on a
log10 scale marked by negative exponents. Values below
1010 were derived from samples in which PML-RAR was
undetectable. To be evaluable, both BM and PB samples had to
have a GAPDH copy number exceeding 2.5 × 105.
Concordance of BM and PB values is indicated by coincident values on
the diagonal, and the magnitude of discordance is indicated by the
distance from the diagonal. The large diamond represents 27 BM-PB pairs
in which evaluable samples from each were NQ.
Thirty-eight NQ+ pairs were also concordant on the
diagonal, while 35 pairs disagreed by 1 log (21 with PB lower than BM;
14 with BM lower than PB); 14 pairs by 2 logs (8 with PB lower than BM;
6 with BM lower than PB); and 4 pairs by 3 logs (4 with PB lower than
BM). In 9 comparisons, BM was positive and PB negative, while in 13 comparisons PB was positive and BM negative. The overall
discordance was 42 pairs with BM greater than PB, and 33 with PB
greater than BM. Neg indicates negative.
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Figure 2 details the number and source of
samples used for analysis, which varied depending on
availability at the 4 monitoring intervals. Figure 2 also illustrates
that the assay sensitivity was variable: maximal for pretreatment and
least for FUP samples (median, 9.1 × 106 and
1.9 × 106 GAPDH copies, respectively). This had an
impact on the ability to detect low levels of MRD in the interval after
consolidation therapy and the FUP interval, since the fraction of
PML-RAR+ samples was reduced in the lower- compared with
the upper-sensitivity range: after consolidation chemotherapy, 30%
versus 47%; FUP, 28% versus 53%. The lower assay sensitivity during
the FUP interval was not due to the higher fraction of substitute PB
samples (20 of 167, 12%) compared with the interval after
consolidation therapy (3 of 53, 6%), since the distribution of GAPDH
and of NQs between PB and BM samples in the FUP was equivalent (data
not shown).
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| Figure 2.
Distribution of evaluable low- and high-sensitivity
range samples and of PML-RAR+ samples at 4 INT0129
monitoring-interval checkpoints.
The columns represent the percentage of evaluable samples (ordinate) in
the higher-sensitivity range (GAPDH exceeding 2.5 × 106
copies;  ) or lower-sensitivity range (GAPDH exceeding
2.5 × 105-2.5 × 106;  ). The percentage
of cases that were PML-RAR+ (NQ+)
in each sensitivity range at the 4 checkpoints is indicated above each
column, while the overall percentage of NQ+ cases is
summarized in the last row at the bottom of the Figure. The first row
at the bottom of the Figure presents the total number of samples at
each checkpoint interval, with the number of PB samples substituted for
BM indicated in parentheses. The median number of GAPDH copies and the
median NQ for NQ+ cases at each checkpoint interval are
indicated in the second and third rows at the bottom of the Figure,
respectively. *During the FUP, the median number of samples per patient
was 2 (range, 1-8).
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Evaluation of PML-RAR NQ for association with pretreatment APL
characteristics
We evaluated NQs for an association with 2 pretreatment
determinations that have been extensively studied for an association with treatment outcome: PML-RAR mRNA type and presenting WBC count.19 We found a highly significant association between
cases with S-form PML-RAR and higher NQs (Table
2). At CR, however, no difference was
observed (P = .457), and in fact, a greater proportion of
S-form cases converted to PML-RAR: 5 (17%) of 29 S-form cases versus 2 (4%) of 47 L-form cases. Similarly, after
consolidation therapy, there was no difference in the distribution
of S- and L-form cases (P = .659) in terms of PML-RAR cases, 9 (56%) of 16 and 22 (61%) of 36, respectively.
In contrast, we found no significant association between pretreatment
WBC count and NQ (P = .808) (Table 2). For this analysis, we asked if there was a difference in NQs for patients with WBC counts
greater than versus less than 2 ×109/L (less than
2000/µL), because this cutoff approximates the median WBC
count (1.7 ×109/L [1700/µL]) for the current study
and because a concurrent re-evaluation of the INT0129 trial indicates
that patients with greater than 2 ×109/L (greater than
2000/µL) have significantly reduced DFS.26 Independent
analyses of evaluable samples from BM (n = 48) or from PB (n = 31)
also showed no association of NQs with WBC count (P = .851, BM; P = .294, PB).
Evaluation of PML-RAR NQ for relation to induction-therapy
response
Of the 123-patient cohort, there were no early deaths, and only 7 patients failed to achieve CR. Pretreatment NQ determinations were
available on 5 of the latter cases (NQ exceeding 0.01, 4 cases; NQ less
than 0.01, 1 case), too few to analyze. Alternatively, we asked whether
NQ at CR correlated with pretreatment NQ, but found no significant
association (n = 42; P = .814). Nor was there a
difference in the time required to achieve CR between the higher and
lower pretreatment NQ groups at the 0.01 cutoff (median times, 45 and
47 days; respectively; P = .586). Additionally, we found no association of pretreatment NQ with 2 complications that occur relatively frequently during the remission induction period in APL,
bleeding/coagulation disorders (P = .163), or, in
ATRA-treated patients, the ATRA syndrome
(P = .619).27-30 Most important, we found no
association between any NQ level, including PML-RAR
versus PML-RAR+, and relapse risk in 61 patients who
were evaluated before treatment and subsequently achieved CR or who, in
76 cases, were evaluated at CR (data not shown).
Another question was whether there was a difference in the clearance of
PML-RAR+ cells on the ATRA- or DA-induction arms of
protocol INT0129. Analysis of 76 CR samples demonstrated significantly
lower NQs for ATRA-treated compared with DA-treated patients
(P = .018; 2-sided Wilcoxon rank-sum test) (Table
3). This included 6 (16%) ATRA-treated
cases versus 1 (3%) DA-treated case with undetectable PML-RAR. The
association of lower NQs with ATRA versus DA induction therapy was also
seen after consolidation therapy (P = .037), at which time
20 (71%) of 28 ATRA-treated versus 12 (48%) of 25 DA-treated patients
were PML-RAR.
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Table 3.
Distribution of PML-RAR NQs after remission induction
with chemotherapy or ATRA at clinical remission and after consolidation
chemotherapy
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Evaluation of PML-RAR NQ after consolidation for relation to
disease-free survival
Evaluable samples were available from 53 patients after
consolidation therapy, 22 (42%) of whom subsequently experienced
relapse over a median follow-up of 69 months. Measurable NQs from these patients were distributed over a greater than 6-log range (lower than
109-103). Table
4 presents the data related to relapse
risk at 3 different NQ cutoff levels, comparing cases with NQs above
the cutoff (poor risk) with those below the cutoff (good risk). The
maximum difference between poor-risk and good-risk cases was
observed at the 105 cutoff level (hazard ratio
[HR] = 4.1; P = .008) (Figure
3). A similar trend was noted at the
107/8 cutoff (HR = 2.4;
P = .074), but there was no significant risk of relapse in
a comparison of PML-RAR versus PML-RAR+
cases (HR = 1.7; P = .244). Notably, 11 (50%) of 22 relapse cases were PML-RAR at the checkpoint after
consolidation therapy (Table 4).
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Table 4.
Hazard ratio and 1- to 3-year DFS analysis at 3 PML-RAR
NQ cutoff levels of samples after consolidation therapy in 53 INT0129 cases
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| Figure 3.
Kaplan-Meier analysis comparing disease-free survival of
patients with PML-RAR NQ exceeding 105 versus patients
with NQ below 105 after completion of protocol INT0129
consolidation therapy.
Open circles indicate censored cases. Median follow-up was 69 months measured from the achievement of complete remission.
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Evaluation of PML-RAR NQ in the follow-up period
The subgroup of patients at the checkpoint after consolidation
therapy with high relapse risk (NQ exceeding 105)
constituted only a minor fraction of evaluable patients (9 of 53; 17%)
and included only about one quarter of cases destined to experience
relapse (6 of 22; 27%). Thus, an important question was whether a
criterion could be established to identify an additional subgroup at
increased risk of relapse in the FUP. For this purpose, 167 evaluable
samples (147 BM plus 20 PB) were available from 70 patients who had a
least 1 sample in the FUP (Figure 2). Each patient was initially
assigned to the good-risk group, but was reassigned to the poor-risk
category at the actual time when the NQ exceeded the specific cutoff
for risk determination. Thus, a patient whose NQ exceeded
106 in a particular FUP sample was considered good risk
before but became poor risk with respect to this cutoff level at the
sample date and remained poor risk even if lower NQs were recorded at subsequent qrtPCR checkpoints. Table 5
illustrates the analysis of poor risk versus good risk for the
possibility of relapse at the 3 most informative NQ cutoff
levels. The analysis was structured in this manner because the data
suggested that there was a substantial difference between the risk of
relapse at the 105 (HR = 17.5) and the
106 (HR = 7.6) cutoff levels, while the HR values at
cutoff levels below 106 (eg, HR = 5.8 for
NQ+ versus NQ) appeared to be almost entirely
driven by the high relapse rate in cases with NQs exceeding
106. As can be easily calculated, of 45 cases with NQs
below 106, relapse occurred in 4 (19%) of 21 cases, with
NQs between greater than 1011 and lower than
106, and in 4 (17%) of 24 NQ cases. These
data suggest that a relatively low and constant risk of relapse is
present at NQ below 106, but that there is a
progressively higher risk at values above this cutoff.
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Table 5.
Hazard ratio and relapse incidence at 3 PML-RAR NQ
levels in follow-up period (FUP) samples from 70 INT0129 cases
after completion of consolidation chemotherapy
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Insufficiencies in the number and regularity of samples did not
permit analysis for a possible relationship of PML-RAR NQs to
clinical outcome in successive samples. Further, serially
monitored NQs in individual cases showed marked heterogeneity of
pattern. In only a minority of FUP cases was CCR associated
with persistent NQ-negativity or was relapse presaged by a distinctive
ascending pattern at the 6-month or longer protocol checkpoint
intervals. Most cases showed a descent to low or undetectable NQs
immediately after consolidation therapy, frequently followed, in cases
with 2 or more samples, by an erratic pattern of various levels of positivity interspersed with negative points. Two examples of such
cases that persisted in CCR, despite intermittent NQ-positivity, are
illustrated in Figure 4. The transient NQ
increases at 12 and 36 months in patient 1 (Figure 4A) or at 6, 12, and
24 months in patient 2 (Figure 4B) are indistinguishable from similar
elevations in other cases that were succeeded by relapse before the
next 6-month checkpoint (eg, Figure 4; Slack et al17). The
data in Figure 4 also illustrate the comparability of PB- and
BM-monitoring values and the qualitative confirmation of low-level
qrtPCR+ NQs by high-sensitivity mrtPCR detection (Figure
4B).
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| Figure 4.
Clinical course and serial monitoring of PML-RAR NQs
derived from bone marrow and/or peripheral blood by real-time
quantitative RT-PCR in 2 patients treated on protocol INT0129.
(A) Patient 1 received induction treatment with daunorubicin plus
cytarabine (DA) and, after 2 courses of DA consolidation chemotherapy,
received all-trans retinoic acid (ATRA) maintenance therapy
for 1 year. (B) Patient 2 received remission induction treatment with
ATRA and, after 2 courses of DA consolidation chemotherapy, received no
further treatment. Both patients are in continuing clinical remission
after longer than 5 years follow-up. The monitoring intervals
designated on the abscissa are representational and not time-linear.
PML-RAR NQs, indicated on the left ordinate, were determined by
dividing PML-RAR copy number by GAPDH copy number. Bone marrow NQs,
stippled columns; peripheral blood NQs, hatched columns. Assay
sensitivity is represented by the reciprocal of the GAPDH copy number
(1 divided by the GAPDH copy number), indicated on the right
ordinate. The sensitivity cutoff at 1/2.5 × 105 GAPDH
copies corresponds to a sensitivity detection level of
104.17 Thus, the further below the cutoff
line, the more sensitive the assay, while values above the line were
considered nonevaluable for use in cohort analyses, whether PML-RAR
NQ or NQ+, although the calculated PML-RAR
NQs for positive samples are included in this chart. Reciprocal GAPDH
values: bone marrow, closed circles; peripheral blood, open triangles.
The presence of these symbols indicates that the sample was assayed; if
the corresponding PML-RAR NQ column is absent and marked with an
asterisk, it means that the sample was NQ. Several bone
marrow and peripheral blood samples for patient 2 were assayed by a
high-sensitivity (hs) mrtPCR assay,17 as well as by
real-time qrtPCR, and ± results are shown below the graph. NT,
not tested.
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Discussion |
In the current study, we investigated the application of
high-sensitivity qrtPCR in a representative subgroup of APL
patients from the phase 3 trial INT0129, which is the first application of this methodology to a large clinical trial study of APL. The data
permit an assessment of the potential prognostic value of qrtPCR at 4 phases of disease relative to protocol therapy: pretreatment; at CR;
after the completion of 2 cycles of consolidation chemotherapy but
before the second protocol randomization (after consolidation therapy);
and during the postconsolidation FUP (median follow-up, 69 months).
The study accomplished the major objective of identifying high-risk
thresholds for disease relapse in the postconsolidation period. At the
checkpoint after consolidation therapy, it was determined that the
subgroup with a PML-RAR NQ exceeding 105 had a
4.1-fold increased risk of relapse, which occurred in 6 (67%) of 9 patients in this category (Table 4; Figure 3). However, these 6 high-risk patients represented only 27% of total relapse cases, which
emphasized the importance of continued monitoring in the FUP to attempt
to identify most relapse cases present after consolidation therapy in
the good-risk subgroup with NQ below 105. Partly related
to sample limitations, this analysis was based on the highest single NQ
achieved by each patient at any time during the FUP. Using time-varying
covariate analysis,23,31 we found that relapse risk was
increased 17.5-fold or 7.6-fold for poor-risk cases at NQ cutoff levels
of 105 or 106, respectively (Table 5).
These cutoffs were associated with relapse rates of 77% for NQ
exceeding 105 and of 42% in the NQ 106 to
105 range, and they identified 43% (NQ exceeding
105) and 65% (NQ exceeding 106) of total
relapse cases. These results strongly suggest that patients with NQ
exceeding 105 after the completion of consolidation
therapy should be considered for further therapy and that patients with
NQ 106 to 105 should be monitored
frequently for a possible increase into the very high-risk range of
greater than 105.
A second important finding in the postconsolidation phase was that
there was no apparent stratification of relapse risk at PML-RAR NQs
below the higher-risk cutoffs. At the checkpoint after consolidation
therapy, relapse occurred in 16 (36%) of 44 patients with NQ below
105 and in 11 (34%) of 32 NQ patients
(Table 4). Similarly, during the FUP, relapse occurred in 4 (19%) of
21 patients in the NQ 1010 to 106 range and
in 4 (17%) of 24 NQ patients (Table 5). Thus, our
PML-RAR NQ evaluation appears to define a single threshold in the NQ
106 to 105 range, above which there is high
risk, and below which there is lower but still significant risk of
relapse. Interestingly, serial monitoring of individual patients
demonstrated that there are frequently marked and sporadic quantitative
variations in NQs within the lower-risk range (Figure 4). At
the relatively infrequent sampling intervals used in this study (6 to
12 months), it was not possible to discern patterns of NQs that
distinguished patients who would continue in CR (Figure 4) from those
who would subsequently experience relapse (Figure 4; Slack et
al17). These observations are consistent with reports that
clinical relapse can occur rather suddenly, within 3 to 6 months, from
low MRD levels, including several reports after negative mrtPCR
assays5,11,12,32 and 7 patients in this study with
negative or very low NQs (below 108). These
considerations argue for more frequent MRD monitoring for 1 to 2 years
after finishing consolidation therapy when relapse risk is highest.
Such monitoring of MRD "kinetics" at short intervals may be able to
detect an upward trend toward the high-risk MRD level, as has been
successfully used, for example, in predicting hematological relapse in
chronic myeloid leukemia.31 Our documentation that
monitoring blood is nearly as effective as monitoring BM could
greatly facilitate in reducing the MRD-monitoring interval to shorter
than 3 months, although this requires more systematic confirmation with
simultaneous blood and marrow testing.
Our observation of frequent sporadic changes in persistent low-level
PML-RAR mRNA levels during continuing CR also has more basic
implications related to the process of leukemia progression. In
addition to confirming that a persistent low level of
PML-RAR+ cells is compatible with prolonged
DFS,18 it suggests that the interplay between residual APL
disease and the host is a dynamic process. In combination with the
observation that clinical relapse not infrequently occurs
rapidly from low MRD levels, it suggests that relapse occurs from a
subpopulation of APL cells that undergoes further change(s) leading to
escape from host control mechanisms. Consistent with this hypothesis,
we have found several INT0129 patients in whom mutations in the
PML-RAR gene emerged only late at the time of clinical
relapse.33 Presumably, clonal escape due to this or other
molecular mechanisms would be reflected in the quantitative rate of
clonal emergence, and thus, another benefit of qrtPCR monitoring may be
to learn more about molecular biological changes that perturb the
leukemic cell-host control balance leading to relapse.
In contrast to results in the postconsolidation period, we did
not find any indication that qrtPCR had prognostic value at earlier
time points in the disease course. At CR, there was no association
between PML-RAR NQ level and DFS (P = .814), and 3 of 7 NQ patients subsequently experienced relapse.
Interestingly, the distribution of NQs at CR was significantly lower in
patients treated with ATRA versus chemotherapy for induction
(P = .018) (Table 4). These findings were unexpected since
previous reports with mrtPCR reported a greater frequency of
PML-RAR assays after chemotherapy versus ATRA
induction therapy.2,8,9 The reason for this difference is
not clear, since INT0129 used a standard ATRA dose (45 mg/m2/d) and schedule and since it was not related to a
difference in the timing of CR and CR sampling (median, 47 and 48 days,
respectively), as identified in a recent report.12 The
greater reduction of NQ by ATRA was also seen after consolidation, when
71% ATRA-treated versus 48% chemotherapy-treated patients were
PML-RAR (Table 4). These observations suggest that the
rate of disease clearance may be an additional quantitative parameter
derived from qrtPCR studies that can be used to assess prognosis and/or the comparative effectiveness of different induction therapies.
We also found no evidence that the pretreatment level of
PML-RAR NQ can add to accepted clinical risk factors in APL,
particularly the presenting WBC count.2,5,19,26,34,35 We
found no association between pretreatment NQ and WBC count or clinical outcome, nor was PML-RAR NQ a predictor for development of
bleeding/coagulation problems and/or the ATRA syndrome during the
induction period. We did find a strong association between high
pretreatment NQ and S-form versus L-form PML-RAR
(P < .001), but this association did not persist after
achieving CR or following consolidation treatment, a finding in
agreement with previous conclusions that PML-RAR type does not
significantly affect the outcome of combined ATRA/chemotherapy
regimens.2,5,19,34,35 A caveat to the conclusion that
pretreatment NQ has no prognostic value relates to our use of RNA
extracted from Ficoll-selected LDMNCs for performing the assays.
Indeed, the lack of association between NQ using RNA from PB samples
and WBC count seems surprising, considering the marked intercase
heterogeneity of APL cell penetration into the vascular compartment at
disease presentation. Thus, we cannot exclude the possibility that NQ
determinations from unfractionated PB leukocytes could have prognostic
significance, although it is unclear if this would add to the value of
WBC count per se.
In summary, this initial monitoring study of APL using qrtPCR indicates
that a significant relationship between the level of PML-RAR mRNA
and clinical outcome was limited to the postconsolidation phase. These
findings are qualitatively similar to those previously reported using
conventional, nonquantitative mrtPCR.5,11,12 By both
methods, the criteria for high risk after consolidation therapy (NQ
exceeding 105 by qrtPCR; PCR+ by
mrtPCR) fail to detect most subsequent relapse cases, but during the FUP (NQ exceeding 105 by qrtPCR; 2 successive
PCR+ assays by mrtPCR) are highly predictive of
relapse. Because of limitations of sample size and of differences in
treatment regimens and sampling schedules, it is not possible to
formally compare the results presented here with those reported in
previous studies using mrtPCR methods. QrtPCR has the theoretical
advantage of providing a precise assessment of the effectiveness of
each test specimen assay. Since, as shown in this study, the assay
sensitivity of evaluable samples can vary more than 40-fold (GAPDH
greater than 2.5 × 105 to greater than 107),
it seems reasonable to assume that results from mrtPCR methods are
subject to similar variations that might affect individual assay results, that is, PCR+ versus PCR,
especially near the sensitivity limit of the mrtPCR assay. Increasing the sensitivity of mrtPCR to attempt to identify patients at a lower risk of relapse would probably diminish the clinical
predictive value of this procedure, since, as we determined by qrtPCR,
low levels of PML-RAR mRNA are not tightly associated with relapse risk. We conclude that further studies using common samples to directly
compare the effectiveness of the 2 methodologies are required to
determine if the theoretical advantage of qrtPCR is of sufficient
practical importance to justify the additional expense of this
high-technology procedure for clinical monitoring of MRD in APL.
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Acknowledgments |
We express our gratitude to the physicians, nurses, data managers,
and patients from ECOG-, SWOG- and CALGB-affiliated institutions for
their cooperation in providing clinical specimens and information for
our investigations. We express our special thanks to Dr David Harrington for his encouragement leading to the completion of this
investigation and to Dr Jerry Radich for critical reading of the manuscript.
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Abstract
Introduction