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Prepublished online as a Blood First Edition Paper on May 31, 2002; DOI 10.1182/blood-2002-04-1130.
CLINICAL OBSERVATIONS, INTERVENTIONS, AND THERAPEUTIC TRIALS
From the Departments of Hematology-Oncology,
Biostatistics, and Pathology, St Jude Children's Research Hospital,
and the University of Tennessee, Memphis.
In children with acute lymphoblastic leukemia (ALL), response to
treatment is assessed by bone marrow aspiration. We investigated whether minimal residual disease (MRD) can be effectively monitored in
peripheral blood. We used flow cytometric techniques capable of
detecting 1 leukemic cell among 10 000 or more normal cells to compare
MRD measurements in 718 pairs of bone marrow and peripheral blood
samples collected from 226 children during treatment for newly
diagnosed ALL. MRD was detected in marrow and blood in 72 pairs and in
marrow but not in blood in 67 pairs; it was undetectable in the
remaining 579 pairs. Remarkably, findings in marrow and blood were
completely concordant in the 150 paired samples from patients with
T-lineage ALL: for each of the 35 positive marrow samples, the
corresponding blood sample was positive. In B-lineage ALL, however,
only 37 of 104 positive marrow samples had a corresponding positive
blood sample. Notably, peripheral blood MRD in these patients was
associated with a very high risk for disease recurrence. The 4-year
cumulative incidence of relapse in patients with B-lineage ALL was
80.0% ± 24.9% for those who had peripheral blood MRD at the end of
remission induction therapy but only 13.3% ± 9.1% for those with
MRD confined to the marrow (P = .007). These results indicate that peripheral blood may be used to monitor MRD in patients with T-lineage ALL and that peripheral blood MRD may provide strong prognostic information in patients with B-lineage ALL.
(Blood. 2002;100:2399-2402) Response to therapy for acute lymphoblastic
leukemia (ALL) is assessed by identifying residual cells that express
morphologic, immunophenotypic, or genotypic characteristics of
leukemia.1 Residual disease investigations at the
completion of remission induction therapy are universally performed on
bone marrow samples. However, the frequency with which residual
leukemia can be monitored is limited, especially in children, by the
discomfort and practical difficulties posed by bone marrow aspiration.
After sensitive methods for minimal residual disease (MRD) detection
became available,2-4 a few investigators recognized the
potential advantage of performing MRD studies on peripheral blood
instead of bone marrow and assessed their value.5-8 It was
found that the detection of MRD in peripheral blood paralleled that in
bone marrow in most patients, but levels of MRD in peripheral blood
were, at most, one tenth of those measured in paired bone marrow
samples. These results suggested the potential usefulness of MRD
studies in peripheral blood, but their interpretation was limited by
the small number of patients in each series and by the lack of
correlative comparisons with presenting features and outcomes.
Moreover, the reported studies included only patients with B-lineage ALL.
Monitoring of MRD in bone marrow samples is known to provide strong
prognostic information about childhood ALL,9-15 and such assays are increasingly used to select the intensity of
treatment.16 We hypothesized that MRD studies in
peripheral blood could also be helpful. ALL of T-cell origin may well
derive from progenitor cells that naturally reside in the thymus rather
than in the bone marrow.17,18 If leukemic T-cell
lymphoblasts migrate to the bone marrow through the circulating blood,
studies of peripheral blood might be as informative as studies of bone
marrow in that subset of patients. In patients with B-lineage ALL
(which originates from bone marrow progenitor cells),18,19
the presence of circulating lymphoblasts at the time of clinical
remission might indicate a propensity of the malignant cells to exit
the bone marrow prematurely. This feature may be associated with
invasion of extramedullary sites, residence in pharmacologic
sanctuaries, and unfavorable outcomes. Here, we present results
suggesting that monitoring MRD in the peripheral blood of children with
ALL may be clinically useful.
Patients
Treatment protocol
Flow cytometric assessment of MRD Aspirated bone marrow and peripheral blood were collected in preservative-free heparin. Leukemia-associated immunophenotypes (combinations of cell markers found on leukemic cells but not on normal bone marrow or peripheral blood cells) were detected by multiparameter flow cytometry with various combinations of monoclonal antibodies and heterologous antisera conjugated to fluorescein isothiocyanate, phycoerythrin, peridinin chlorophyll protein, and allophycocyanin.15,21,24 Matched, nonreactive, fluorochrome-conjugated antibodies served as controls. The staining procedure has been described.24,25 For each patient, one or more marker combinations that allowed the identification of 1 leukemic cell in 104 or more normal nucleated bone marrow or peripheral blood cells as determined by testing samples from
healthy donors, patients undergoing treatment, and artificial mixtures
of leukemic and normal cells24,26were selected at the
time of diagnosis and applied during treatment. In the early part of
the study, we used 3-color analysis with a single-laser FACScan flow
cytometer; after August 1998, all samples were assayed by 4-color
analysis and processed with a dual laser FACSCalibur flow cytometer
(both cytometers were from Becton Dickinson, San Jose, CA). The flow
cytometry protocol used for MRD detection has been described in detail
previously.24,25 In each test of each sample, we acquired
data from more than 105 mononuclear cells. Detectable MRD
was defined as 0.01% or more cells expressing a leukemia-associated
immunophenotype among mononuclear cells in the sample.
Statistical analysis Exact 2 analysis and Fisher exact test
were used to compare differences in the distribution of clinicobiologic
presenting features at the end of remission induction therapy for
B-lineage ALL according to the presence of MRD in peripheral blood. The cumulative incidence of relapse of ALL in patients treated on earlier
protocols with adequate follow-up was calculated by the Gray method,
adjusting for other competing risks (ie, second malignancy and death
during remission). The cut-off date for follow-up observations was
February 2002. Patients who underwent hematopoietic stem cell transplantation were censored at the time of relapse, a competing event, or last follow-up date.
In 139 (19.4%) of the 718 bone marrow samples studied, 0.01% or more of the mononuclear cells expressed the leukemia-associated immunophenotype determined at diagnosis. Levels of residual disease ranged from 0.01% to 53.21% (median, 0.20%). Of the corresponding 139 peripheral blood samples, 72 (51.8%) contained 0.01% or more cells with the leukemic phenotype, whereas 67 (48.2%) contained no leukemic cells detectable by flow cytometry (less than 0.01%). In the peripheral blood samples that were positive for MRD, findings ranged from 0.01% to 50.04% (median, 0.05%). The remaining 579 bone marrow samples were MRD-negative; all of their paired peripheral blood samples were MRD-negative. The distribution of residual disease differed markedly between patients
with T-lineage ALL and patients with B-lineage ALL. In patients with
T-lineage ALL, detectable MRD in bone marrow was consistently
accompanied by detectable MRD in peripheral blood, at all time points.
Of the 150 pairs of samples from patients with T-lineage ALL, 35 (23.3%) showed detectable MRD in bone marrow and blood, and the paired
samples generally had similar proportions of leukemic cells (Figure
1). The Pearson correlation coefficient was 0.8183, and, by regression analysis, the slope of the regression line was not significantly different from that of the line of unity
(P = .703). In only 5 of the 35 (14.3%) sample pairs was the level of MRD in the bone marrow sample more than 10 times that in
the blood sample, and, in one pair, MRD in the blood sample was more
than 10 times that in the marrow sample. Therefore, in T-lineage ALL,
the proportion of MRD in peripheral blood generally reflected that in
the bone marrow.
In patients with B-lineage ALL, MRD was more prevalent in bone marrow than in peripheral blood. Of the 568 pairs of B-lineage ALL samples, 104 (18.3%) had detectable MRD in the bone marrow, but only 37 of these had detectable disease in the corresponding peripheral blood sample, a result significantly different from that obtained in T-lineage ALL, where all 35 bone marrow samples with detectable MRD had detectable disease in the corresponding blood sample (P < .001, Fisher exact test). Moreover, the level of MRD in blood was generally lower than that in the paired bone marrow sample (Figure 1). The Pearson correlation coefficient was 0.4162, and, by regression analysis, the slope of the regression line was significantly different from that of the line of unity (P = .012). In 24 of the 37 sample pairs in which the peripheral blood was positive (64.9%), the bone marrow sample had a level of MRD more than 10 times as high; the reverse did not occur. Of the 47 MRD-positive marrow samples collected during clinical remission, 9 had a paired blood sample that was MRD-positive. The presence of MRD in the blood was generally associated with higher levels of MRD in marrow. The 9 patients who had peripheral blood MRD had a median of 0.7% MRD in the marrow (range, 0.02%-3.3%), whereas the 38 patients who had MRD only in the marrow had a median of 0.05% MRD (0.01%-3.8%). However, levels of MRD in the marrow in the 2 groups largely overlapped, and the presence of MRD in blood was not limited to patients with high MRD in the marrow. This finding suggested that MRD in the peripheral blood is dependent on specific biologic features of the leukemic cells rather than on physical disruption of the blood-bone marrow barrier. We assessed the relationship between the presenting cellular and
biologic features of patients with B-lineage ALL and the presence of
MRD in peripheral blood at the end of remission induction therapy (day
46). Of the 171 patients studied at this time point (all in clinical
remission), 41 had detectable MRD in the bone marrow and 8 of the 41 also had detectable MRD in the blood. We detected no obvious
association among the presence of MRD in the peripheral blood and
leukemic cell ploidy, rearrangement of the TEL or
MLL genes, presence of the BCR-ABL fusion gene,
or age, sex, race, or leukocyte count at diagnosis, though the
low number of patients studied limited the power of the statistical
analysis. We also found no apparent relationship between MRD in
peripheral blood and previous treatment with G-CSF. We then
investigated whether the presence of MRD in the peripheral blood could
identify patients at higher risk for relapse. We restricted the
analysis to 65 patients with B-lineage ALL who were treated on earlier protocols, had been followed for at least 2.5 years, and had not undergone risk-adapted therapy based on MRD findings. All 65 patients were in clinical remission at the end of remission induction therapy (day 46), but 20 had detectable MRD (0.01% or more) in the bone marrow
at that time. Five of these 20 patients also had detectable MRD in the
peripheral blood. We found no apparent differences in the morphologic,
immunophenotypic, or karyotypic features of the malignant cells or in
clinical presenting features between patients who had MRD only in the
bone marrow and those who had MRD in bone marrow and peripheral blood.
The 4-year cumulative incidence of relapse was 7.4% ± 4.2% for the
45 patients with no detectable MRD and 32.9% ± 12.0% for the 20 with detectable MRD (P = .019). Remarkably, 4 of the 5 patients with detectable MRD in the peripheral blood had relapses
compared with only 2 of the 15 patients with MRD only in the bone
marrow (4-year estimated cumulative incidence of relapse,
80.0% ± 24.9% vs 13.3% ± 9.1%; P = .007) (Figure
2).
We found that the distribution of MRD differs radically in patients with T-lineage and B-lineage ALL. In patients with T-lineage ALL, cells expressing a leukemia-associated immunophenotype were consistently present in the peripheral blood when they were detected in the bone marrow. Further, their proportions in peripheral blood were remarkably similar to those obtained in the bone marrow, irrespective of the time point at which they were measured. This finding suggests that peripheral blood could be used for MRD studies in patients with T-lineage ALL and warrants further study in patients enrolled in different treatment protocols, monitored at different intervals, or both. Use of peripheral blood would allow closer monitoring of leukemia while sparing patients the discomfort of bone marrow aspirations. In sharp contrast to findings in patients with T-lineage ALL, those
with B-lineage ALL often had no detectable cells with leukemia-associated immunophenotypes in peripheral blood, despite their
presence in bone marrow. This result is consistent with those of
previous studies of MRD in peripheral blood, which have focused on
patients with B-lineage ALL. Brisco et al5 used polymerase
chain reaction (PCR) amplification of immunoglobulin genes to quantify
MRD in 35 pairs of bone marrow and blood samples from 15 children
receiving remission induction therapy. They found that the levels of
MRD in blood were lower than those in bone marrow by a factor of 10. Van Rhee et al6 used reverse transcription-PCR (RT-PCR)
amplification to study p190 BCR-ABL transcripts in 29 pairs
of samples from 18 patients receiving treatment for Philadelphia chromosome-positive B-lineage ALL. As a whole, the numbers of BCR-ABL transcripts detected in peripheral blood were not
significantly different from those detected in bone marrow. However,
MRD was detectable in the bone marrow but not in the blood in 4 sample pairs; in 3 additional pairs, MRD was detected in both samples but was
more than 10 times as high in marrow than in blood. Using the same
approach, Martin et al7 studied 9 pairs of samples from 6 patients and found that MRD levels in marrow exceeded those in blood by
a factor of 10 or more in every case. These findings, taken together
with our results, clearly indicate that in B-lineage ALL, levels of MRD
in bone marrow are usually higher than those in peripheral blood.
However, in our series, the presence of MRD in peripheral blood denoted
more aggressive leukemia with an extremely high risk for recurrence
We thank Peixin Liu and Mo Mehrpooya for technical assistance, Yinmei Zhou for assistance with the statistical analysis, Geoffrey Neale for helpful discussions, and Sharon Naron for editorial suggestions.
Submitted April 15, 2002; accepted May 16, 2002.
Prepublished online as Blood First Edition Paper, May 31, 2002; DOI 10.1182/blood-2002-04-1130.
Supported by grants CA60419, CA21765, and CA20180 from the National Cancer Institute, by the Rizzo Memorial Grant from the Leukemia Research Foundation, and by the American Lebanese Syrian Associated Charities.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Dario Campana, Department of Hematology-Oncology, St Jude Children's Research Hospital, 332 N Lauderdale, Memphis TN 38105-2794; e-mail: dario.campana{at}stjude.org.
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C. Flotho, E. Coustan-Smith, D. Pei, S. Iwamoto, G. Song, C. Cheng, C.-H. Pui, J. R. Downing, and D. Campana Genes contributing to minimal residual disease in childhood acute lymphoblastic leukemia: prognostic significance of CASP8AP2 Blood, August 1, 2006; 108(3): 1050 - 1057. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
), at the end of remission induction (day 46; 
), and
during weeks 14, 32, 56, or 120 of continuation chemotherapy (
). In
the lower left quadrant, numbers of MRD-negative paired samples
collected at the various treatment intervals are shown.
). Solid lines indicate the
cumulative incidence of relapse of MRD+ patients with MRD confined to
the bone marrow (BM) or detectable MRD in the peripheral blood
(PB).