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CLINICAL OBSERVATIONS, INTERVENTIONS, AND THERAPEUTIC TRIALS
From the Children's Cancer Research Institute, St Anna
Kinderspital, Vienna, Austria.
Detection of minimal residual disease (MRD) in acute lymphoblastic
leukemia (ALL) predicts outcome. Previous studies were invariably based
on relative quantification and did not investigate sample-inherent
parameters that influence test accuracy, which makes comparisons and
clinical conclusions cumbersome. Hence, we conducted a prospective,
population-based MRD study in 108 sequentially recruited children with
ALL uniformly treated with the ALL-Berlin-Frankfurt-Münster
(ALL-BFM) 95 protocol in Austria (median follow-up of 40 months). Using
sensitive, limited antibody panel flow cytometry applicable to 97% of
patients, we investigated 329 bone marrow samples from 4 treatment time
points. MRD was quantified by blast percentages among nucleated cells
(NCs) and by absolute counts (per microliter). Covariables such as NC
count, normal B cells, and an estimate of the test sensitivity were
also recorded. Presence and distinct levels of MRD correlated with a
high probability of early relapse at each of the time points studied.
Sequential monitoring at day 33 and week 12 was most useful for
predicting outcome independently from clinical risk groups: patients
with persistent disease ( Measurement of minimal residual disease (MRD) by
flow cytometry (FC) or polymerase chain reaction (PCR) emerges as an
attractive new tool for risk assessment in childhood acute
lymphoblastic leukemia (ALL).1 Several studies with FC,
which bears the methodologic advantage of being relatively simple and
quick, have demonstrated that MRD detection based on
leukemia-associated phenotypes correlates with outcome and should be
applicable to a majority of children with ALL.2-6 Two
large FC investigations established the significance of different
levels of MRD at sequential time points in therapy, proving that MRD is
an independent outcome indicator.7-9 Between these studies
and in comparison to the most relevant PCR-derived data,10,11 divergences exist regarding the proportion of
MRD+ patients at distinct treatment time points, the
prognostic relevance of certain MRD levels, and the general
applicability of techniques that compete in the quest for the most
favorable expenditure/efficacy profile. Because quantitative data
generated by PCR and FC seem largely interchangeable,12 it
needs to be determined in the future whether these divergences are
mainly caused by differences between therapeutic regimens or by
technology. Notably, all studies published to date have relied on only
relative measurements of MRD (relative to leukemic genome copy
standards or to cells of a sample) mostly among mononuclear cell
preparations. It is therefore tempting to speculate whether
methodologic standardization on the basis of truly absolute
quantification of MRD among total nucleated cells (NCs) in combination
with a better understanding of the dynamic changes of cell content and
cellular composition of the bone marrow (BM) during treatment provides
even more accurate and prognostically reliable information.
Hence, we conducted a prospective FC study that allowed investigation
of the values of relative and absolute MRD quantification as well as
the modalities of assessment in pediatric ALL patients with
risk-directed therapy. Our method was based on a limited antibody panel
approach applicable to most patients due to a combination of techniques
directed toward investigation of B-cell precursor (BCP) and T-lineage
phenotypes, and enabled the detection of one leukemic cell among at
least 104 normal cells.4,5,13-18
Patients and samples
Treatment protocol
Sample preparation and immunofluorescence staining Heparinized BM samples were usually received by express mail within 1 day after collection and immediately processed. First, the NC count of each aspirate was assessed with a Sysmex F-820 cell counter (TOA Medical Electronics, Hamburg, Germany). Subsequently, NCs were prepared using a commercially available red cell lysing solution (Becton Dickinson, Sunnyvale, CA). The panel of monoclonal antibodies (MoAbs) for immunofluorescence staining has been delineated,4,13,21,22 except for the following unconjugated or fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)-, peridinin chlorophyll protein (PerCP)-, PE-cyanin 5.1 (PC5)-, or allophycocyanin (APC)-labeled MoAbs: CD3 (UCHT1-FITC,-PE, pure), CD20 (B-Ly1-PE), and CD38 (AT13/5-FITC) from Dako (Glostrup, Denmark); CD19 (SJ25C1-APC), and CD45 (2D1-PerCP) from Becton Dickinson; CD34 (581-PC5) from Beckman-Coulter (Vienna, Austria). Except for investigations of normal BCPs, which were done with 4 directly labeled MoAbs, the panel was based on the use of 3-color stainings including one unconjugated MoAb. The labeling cascade, quality control measures, and the cellular permeabilization procedure have been described in detail.4,21,22FC analyses Analyses were performed on a dual-laser FACSCalibur (Becton Dickinson). Details regarding the test standardization, the data acquisition with the CELL Quest software (Becton Dickinson), and the data analysis using the PAINT-A-GATE software (Becton Dickinson) have been delineated.4,13,22 We used several standardized antibody combinations to screen ALL samples at diagnosis for leukemia-associated aberrations as well as to investigate follow up BM.5 First-line strategic MoAb combinations against frequently aberrant antigens were used in all patients: for BCP-ALL CD34/CD10/CD19 (in pro-B ALL: CD34/CD10 plus CD20/CD19) and CD10/CD45RA/CD19, and for T-ALL TdT/cytoplasmicCD3/surfaceCD3 as well as cytoplasmicCD3/CD7/surfaceCD3. In the first 72 sequential patients, first-line combinations were always complemented in case of BCP-ALL by additional MoAb triplets (CD10/CD45RA/CD11a, CD10/CD45RA/CD44, CD10/CD33/CD19, and cytoplasmic IgM/CD34/CD19), which were used in follow-up investigations whenever applicable. In later cases (n = 36), full typing including the complementary markers was only done if aberrations relevant for follow-up could not be determined with the first-line marker panel (n = 8). Aberrant characteristics were judged relevant only if they were sufficiently strong or homogeneous on a majority of blasts of a leukemia sample. Table 1 summarizes the leukemia-associated characteristics used to monitor MRD in the 108 ALL cases. Fifty-two leukemias had one aberrant marker (48.1%), 53 (49.1%) had more than one (39 had 2, 11 had 3, and 3 had 4), and only 3 (2.8%) did not have a useful aberration.
Stainings with the cell-permeant, live-cell nucleic acid fluorochrome
SYTO 16 (emission at 518 nm; Molecular Probes, Leiden, The Netherlands)
combined with CD19 or CD3/CD45 were used in follow-up analyses to
exclude residual nonnucleated erythroid cells, thrombocytes, or debris
(SYTO16 Data handling Minimal residual disease was defined as accumulation of events with leukemia-associated phenotypic characteristics in follow-up BM. A relative estimate (percentage of leukemic cells among NCs) and an absolute count (leukemic cells per microliter) were assessed. Absolute counts were calculated by multiplying blast percentage values with the total NC count of the sample. Events that did not form typical cellular clusters within the regions of FC parameter correlations expected to contain only leukemic cells, or that rather overlapped leukemia-associated regions from adjacent areas, were regarded indefinite but similarly quantified. Such events occurred as infrequent background related to diminished sample quality or as normal populations, which to some extent resembled leukemic cells by phenotype (eg, immature normal BCP). Samples containing only such indefinite events were judged MRD , but the proportion and absolute
number of indefinite events was recorded as threshold of sensitivity,
which reflected that MRD more numerous than this value did not exist in
a given sample. Because occurrence of such indefinite events did not
preclude that leukemic cells, if present, could accurately be
discerned, unfavorably high thresholds were not considered an
obligation to exclude samples from further analyses. Rather, respective
data were analyzed with respect to the prognostic reliability of the test system as well as their association with sample composition parameters and time points.
Statistical analysis The Wilcoxon 2-sample test was used to investigate for differences in sample composition parameters and sensitivity thresholds. Associations between these data were assessed with the Spearman rank correlation. Differences between patient groups regarding the proportion as well as the log-level distributions of MRD+ samples were estimated using the Mantel-Haenszel 2 test. The principal end point used to determine the
prognostic value of MRD results was the relapse-free interval (RFI),
calculated as the interval from the time point of a given assessment
until the date of first relapse or end of observation. Four patients who underwent BM transplantation (including the 2 patients who failed
to attain remission at day 33) and did not relapse thereafter, and 3 patients who died from causes other than leukemia itself (infection or
thromboembolism), were included but censored with the date of
transplantation or death. The probability of the RFI (pRFI) was
estimated by the method of Kaplan-Meier.25 SDs of Kaplan-Meier estimates were calculated according to Greenwood. The
log-rank test was used to explore the prognostic impact of MRD in
univariate analyses. A Cox regression model, adjusted for BFM risk
groups, was used for multivariate analyses.26 Relative hazard rates (CI 95%) were calculated, a Wald test was used to test
the significance of differences between groups, and the predictive value of MRD results additional to BFM risk groups was examined by the
log-likelihood ratio test.
MRD results and influencing factors We assessed MRD on the basis of relative and absolute blast cell measures in children with ALL (Table 2). Follow-up BM samples (n = 329) were taken at 4 standardized time points during the first 6 months of treatment. Covariables such as the NC count, the total normal B-cell content, and the BCP subset composition were also investigated. The proportion of samples that were MRD+ was high at day 15 (89.1%), and decreased thereafter to 40.9% at day 33, 13.7% at week 12, and 4.3% at weeks 22 to 24 (Table 2). The MRD burden of positive samples is reviewed in Table 2.
Total NC counts showed a high degree of variance between individual
samples of a given time point as well as between time points in general
(Table 3; P < .001 for the
differences between time points except for week 12 versus weeks 22 to
24, which was not significant). The lowest values were usually found at
day 15, and the highest at week 12 and weeks 22 to 24. Related to the
variance in NC counts, absolute blast counts were classified to
different log-levels compared to the respective relative estimates in
59 (38.8%) of 152 MRD+ samples. The composition of BM
samples regarding normal BCP subsets changed also significantly during
follow-up (Table 3). Samples from day 15 and day 33 contained almost
exclusively mature B cells (stage 3). By contrast, samples from week 12 and weeks 22 to 24 were dominated by very immature BCP (stage 1;
P < .001 for the differences between the earlier and the
later time points), which are phenotypically related to leukemic cells.
The relative proportion of total normal B cells among NCs regressed
from day 15 to week 12, and slightly increased thereafter (weeks
22-24). Related to these peculiarities of the B-cell pool, the
proportional sensitivity threshold values of samples judged
MRD
Outcome and correlation with MRD results Thirteen of the 108 study patients had relapse during the observation period. Presence of MRD was associated with a greater likelihood of relapse at day 33 (P = .024) as well as at week 12 and weeks 22 to 24 (P < .001 for both correlations), but not at day 15. More importantly, the incidence of relapses correlated with distinct levels of MRD positivity. As shown in Table 4, the prognostically relevant levels of MRD decreased with time under treatment (day 15: 1.0% of
NC or 100 blasts/µL BM; day 33: 0.1% or 10/µL; week
12: 0.01% or 1/µL; weeks 22-24: any MRD positivity). Of
note, SR patients, who received a slightly less intensive induction
treatment, did not differ from MR patients regarding the proportion of
positive samples and the distribution among levels of MRD both at day
15 and day 33. Figure 1 summarizes the
predictive value of MRD results at each time point. The accuracy in
defining patients with relapse culminated at week 12 by continuously narrowing the population at risk. Low or absent MRD did not allow a
further substratification with respect to favorable therapy outcome.
Including BFM risk groups in a multivariate Cox model, quantitative MRD
results proved to be independent prognostic factors at day 15 (only
absolute estimate), and in particular at day 33 and week 12 (both
estimates, Table 5). At weeks 22 to 24, too few data were available for these calculations.
Strategic combination of day 33 and week 12 MRD results Ninety-nine percent of MRD tests at day 33 (102 of 103 samples) had a sensitivity that included the levels relevant for outcome prediction at this time point, whereas 70% of assessments (45 of 64) at week 12 had a reduced sensitivity according to threshold proportions. Invariably, patients with predicted relapses had high MRD loads both at day 33 and week 12. To neutralize the surmised reduced reliability of week 12 assessments, we found it useful to combine MRD information from day 33 (for a sensitive predefinition of patients at risk) and week 12 (for the confirmation of an adverse outcome based on the kinetic evolution of MRD between the 2 time points). The predictive value of the strategic combination of the 2 time points is shown in Figure 2. Patients were split up by MRD results into 2 groups with greatly different prognosis (P < .001). Of note, 68 measurements at week 12 were available (n = 51 from consecutive patients; patients recruited later during the study were tested only if MRD+ at day 33, ie, n = 17). Data of a further 34 children were imputed because MRD at day 33 into the MRD low-load group of the combined
analysis, because only 2 of 208 paired assessments from 2 consecutive
time points displayed an increase in MRD. One sample-pair showed
increasing MRD positivity preceding a very early relapse. The other was
found 0.001% positive by week 12, although previously and thereafter judged MRD. All other 78 sample-pairs, which were
negative at the earlier time point, were negative also at the following
investigation. Data of further 1 (absolute count) and 2 patients
(relative estimate), respectively, with high a MRD load on day 33 but
lacking week 12 results were imputed in the MRD high-load cohort.
Patients with adverse outcome (pRFI 0.0 at 3 years) were those with an MRD at least 0.1% of NC (or 10 blasts/µL BM) at day 33, who remained MRD+ at least 0.01% (or 1 blast/µL) at week
12. All other patients had a pRFI of 0.93 (± 0.03 SD) according to
the relative MRD estimate, and of 0.94 (± 0.03 SD) with absolute
counting. In multivariate analysis (Cox model), MRD-based risk
assignment was found to be an independent and overriding prognostic
factor, whereas conventional BFM risk grouping had no significant
additional prognostic impact (log-likelihood ratio-test
P < .001; Table 5). BFM HR patients were split up by
MRD-based stratification into a group that relapsed (pRFI 0.0 at 3 years; n = 8) and a group (n = 8) that profited from chemotherapy
alone (pRFI 0.75 ± 0.15 by relative estimate, P = .018;
0.86 ± 0.13 by absolute count, P = .004). Of note, 1 of
2 BFM HR patients with a relapse despite favorable relative MRD results
had a localized disease recurrence in the CNS. The other patient was
correctly assigned a high risk by the absolute MRD count. Of the 6 BFM
MR patients with a relapse, 2 were predicted by MRD results. The
subgroup of BFM HR patients with a poor response to the prednisone
prephase (n = 9) was also split up by MRD results. Patients with
continuously high MRD load (n = 4) relapsed in 3 (1 censored due to
transplantation) and 4 cases (relative versus absolute estimate),
respectively, whereas of patients with low MRD (n = 5) only 1 patient
suffered a relapse in the relative assessment group,
P = .024, and none in the absolute count group, P = .008. Notably, only 1 of these 9 prednisone poor
responders had an additional HR criterion, that is, a t(4;11).
Analyses of relapse patients In all 10 investigated relapse cases, the leukemic phenotype was stable compared to primary diagnosis in at least one marker used for MRD assessment. BM samples (n = 11) from aspirations preceding these relapses were available in 9 patients (6 MRD+, 5 MRD). The median interval between these aspirations and
relapse diagnosis was 3 months in positive cases (range, 0.5-6 months),
and 6 months in negative cases (range, 4-16 months). Three of 5 patients with MRD+ results had been negative in prior
follow-up investigations, whereas 2 patients had been continuously
positive for 5 and 7 months since primary diagnosis.
Sequential monitoring of MRD during the first months of therapy
provides information on the timely response to treatment and proves to
be a powerful and independent indicator of treatment outcome in
children with ALL.7-11 Our population-based prospective study on 108 unselected pediatric ALL patients, which were uniformly treated according to the widely used ALL-BFM 95 protocol, confirms and
extends these observations. We used an FC method based on a limited
panel of antibodies that was applicable to 97% (105 of 108) of
analyzed patients. At any time point, presence of MRD was associated
with a higher risk of disease recurrence. Distinct cutoff levels of MRD
were found, which correlated with a particularly high relapse hazard,
and which declined with time in therapy. At day 33, patients at risk
could sensitively be predefined (cutoff In several aspects our FC data confirm those generated with PCR
technology by van Dongen et al11 on the basis of a related regimen (protocol ALL-BFM 90); however, some divergences exist. Those
between the prognostically relevant levels of MRD at week 12 (10 Differences in intensity and schedule of therapeutic regimens may also evoke divergences in results and in predictive value of MRD assessment. This seems corroborated by our data that show a decline of the prognostically relevant MRD cutoff over time correlated with the cumulative exposure to cytotoxic drugs. In line with this, markedly different percentages of patients MRD+ at high levels after induction have been observed with 2 different therapeutic regimens that bear similar cure expectancies.28 It may therefore be necessary that MRD data for outcome prediction are generated separately for each therapeutic regimen. In our study, we quantified residual leukemia on the basis of total NCs, which bears conceptual advantages regarding test accuracy over the common practice of analyzing mononuclear cell preparations. By our procedure, assessments can be corrected for bias otherwise caused by variations in preparative quality (increased amount of residual nonnucleated events, eg, erythrocytes, and diminished efficacy in separation of mononuclear cells by imbalance of anticoagulants or prolonged storage), although the theoretical possibility of sample dilution by blood remains a factor that still could adversely influence any type of assessment. However, based on NC analysis, truly absolute MRD quantification by concept may further allow us to correct for a bias that occurs in relative investigations due to differences in total cell content when comparing a series of individual specimens from a given time point. Such differences in NC counts of follow-up samples precipitated log-level divergences in 38.8% of relative/absolute measurement pairs, which led to several divergences also in risk estimates. Day 15 results (only the absolute MRD estimate allowed a statistically significant separation of patients), odds ratios of multivariate analyses, as well as data from individual patients (eg, one patient who relapsed was defined as HR by absolute MRD assessment only) suggested that absolute assessment allows an even more precise risk estimation than relative measurement by ranking samples with high relative MRD values but low total NC counts prognostically more correct. Associated with the individual kinetics both of cell depletion with cytotoxic therapy and, later on, of hematopoietic regeneration after rest from chemotherapy, we found that NC counts differed largely also between samples from different follow-up time points of an individual patient. Therefore, relative measures may also not allow us to reliably assess the quantitative evolution of leukemia over time. In this respect, we found generally higher NC counts as well as a completely different BM composition regarding normal BCP at the 2 later time points compared to the former, which extends recent observations.21,29,30 Our data substantiate speculations that variations in the occurrence of immature lymphoid precursors, which resemble leukemic cells by phenotype, may influence MRD detection.3,6,21 Regenerative time points (week 12 and weeks 22-24) were characterized by a limited sensitivity in MRD assessment in a high proportion of B-lineage cases due to the preponderance of resurgent normal very immature BCP, whereas the high reliability of testings at earlier time points was based on the exclusive presence of mature B cells (apart from residual leukemic cells). Hence, options for investigating MRD may not only depend on leukemia itself, but more importantly also on time point-related factors such as total count and developmental stage of normal cells. Strategic placement of assessments into nonregenerative phases of treatment may thus allow an accurate and sensitive determination of MRD although using a single marker combination that is sufficiently broad to reach most patients. Reducing the need for a diversified investigative setup should add to the clinical significance of FC also by augmenting its economic efficiency. We suggest that a methodologic standardization in these respects is a prerequisite for drawing reliable conclusions for clinical decision making on the basis of MRD.
This study is dedicated to Professor Hansjörg Riehm who pioneered the usage of treatment response assessment for risk estimation in childhood ALL. We thank the participants of the Austrian BFM Study Group (B. Ausserer, F.M. Fink, H. Haas, N. Jones, R. Jones, W. Kaulfersch, G. Müller, I. Mutz, R. Ploier, K. Schmitt, O. Stöllinger, and C. Urban) for their close collaboration in providing BM samples as well as clinical data. In this respect, we are also grateful to the hemato-oncology and the laboratory teams of the St Anna Kinderspital (in particular to S. Juhasz, R. Kornmüller, E. Neidhart, and U. Stalze). We thank J. Regelsberger from the Austrian BFM Study Group documentation center for support, and P. Buchinger, D. Scharner, as well as D. Wimmer for excellent technical assistance.
Submitted April 2, 2001; accepted November 2, 2001.
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: Michael N. Dworzak, Children's Cancer Research Institute, St Anna Kinderspital, Kinderspitalgasse 6, A-1090 Vienna, Austria; e-mail: dworzak{at}ccri.univie.ac.at.
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© 2002 by The American Society of Hematology.
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M. J. Borowitz, M. Devidas, S. P. Hunger, W. P. Bowman, A. J. Carroll, W. L. Carroll, S. Linda, P. L. Martin, D. J. Pullen, D. Viswanatha, et al. Clinical significance of minimal residual disease in childhood acute lymphoblastic leukemia and its relationship to other prognostic factors: a Children's Oncology Group study Blood, June 15, 2008; 111(12): 5477 - 5485. [Abstract] [Full Text] [PDF]
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J. Zhou, M. A Goldwasser, A. Li, S. E. Dahlberg, D. Neuberg, H. Wang, V. Dalton, K. D McBride, S. E. Sallan, L. B Silverman, et al. Quantitative analysis of minimal residual disease predicts relapse in children with B-lineage acute lymphoblastic leukemia in DFCI ALL Consortium Protocol 95-01 Blood, September 1, 2007; 110(5): 1607 - 1611. [Abstract] [Full Text] [PDF]
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D. Hoelzer, N. Gokbuget, O. Ottmann, C.-H. Pui, M. V. Relling, F. R. Appelbaum, J. J.M. van Dongen, and T. Szczepanski Acute Lymphoblastic Leukemia Hematology, January 1, 2002; 2002(1): 162 - 192. [Abstract] [Full Text] [PDF]
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Abstract
Introduction
