Minimal Residual Disease Detection in Childhood Precursor–B-Cell Acute Lymphoblastic Leukemia: Relation to Other Risk Factors

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Leukemia (2003) 17, 1566–1572 & 2003 Nature Publishing Group All rights reserved 0887-6924/03 $25.00 www.nature.com/leu Minimal residual disease detection in childhood precursor–B-cell acute lymphoblastic leukemia: relation to other risk factors. A Children’s Oncology Group study MJ Borowitz1, DJ Pullen2, JJ Shuster3,4, D Viswanatha5, K Montgomery6, CL Willman5 and B Camitta7

1Johns Hopkins Medical Institutions, USA; 2University of Mississippi, USA; 3University of Florida, Gainesville FL, USA; 4Children’s Oncology Group, Arcadia, CA, USA; 5University of New Mexico, USA; 6Genzyme Genetics, Santa Fe, NM; and 7Milwaukee Childrens Hospital, USA

Minimal residual disease (MRD) can be detected in the marrows prognostic significance at several time points. MRD levels at the of children undergoing chemotherapy either by flow cytometry end of induction are highly prognostic. Since intensification of or polymerase chain reaction. In this study, we used four-color therapy after successful induction can improve the outcome of flow cytometry to detect MRD in 1016 children undergoing 33 therapy on Children’s Oncology Group therapeutic protocols many patients with adverse prognostic features, it is possible for precursor–B-cell ALL. Compliance was excellent, with that a similar approach might benefit patients with MRD. For follow-up samples received at the end of induction on nearly these reasons, we have chosen to focus our MRD studies on 95% of cases; sensitivity of detection at this time point was at end-of-induction therapy. least 1/10,000 in more than 90% of cases. Overall, 28.6% of Although MRD and other measures of early response to patients had detectable MRD at the end of induction. Patients 34–36 with M3 marrows at day 8 were much more likely to be MRD therapy are important to prognosis, many other clinical and positive (MRD+) than those with M2 or M1 marrows. Different biological factors also affect outcome. Chromosome number genetically defined groups of patients varied in their prevalence and certain translocations also identify patients with good or of MRD. Specifically, almost all patients with BCR-ABL had high poor prognoses.37–41 Most studies of MRD have not had enough levels of end-of-induction MRD. Only 8.4% of patients with TEL- patients to determine the relation between the presence of AML1 were MRD+40.01% compared with 20.3% of patients with residual leukemia and these other factors. Moreover, although trisomies of chromosomes 4 and 10. Our results show that MRD MRD has been shown to be an independent prognostic factor in correlates with conventional measures of slow early response. 1,9,14,30 However, the high frequency of MRD positivity in favorable multivariate analysis, the precise relation between trisomy patients suggests that the clinical significance of MRD morphologic assessment of slow early response and MRD has positivity at the end of induction may not be the same in all been studied incompletely. In this report, we describe the patient groups. relation between the end-of-induction MRD as detected by Leukemia (2003) 17, 1566–1572. doi:10.1038/sj.leu.2403001 multiparameter flow cytometry and other known prognostic Keywords: acute lymphoblastic leukemia; minimal residual disease; flow cytometry factors, in a series of more than 1000 children with precursor-B- cell ALL. We find that there is a significant correlation between end-of-induction MRD and day 8 morphologic assessment of marrow response. In addition, we find significant differences among MRD rates in genetically defined groups of patients, Introduction which suggest that the clinical significance of MRD at the end of induction may not be the same in all patient subgroups. Minimal residual disease (MRD) can be detected in many patients, who undergo therapy for acute lymphoblastic leukemia (ALL). 1–16 MRD may be detected at a sensitivity of at least 10À4 Materials and methods either by molecular means, generally based on polymerase chain reaction (PCR) amplification of rearranged immunoglo- bulin (Ig) or T-cell receptor genes13,14,16–20 or by multiparameter Patient samples flow cytometry.1,2,8–10,15,21–26 The latter approach is based on The children in this study were enrolled on one of three ongoing the fact that leukemic cells express aberrant phenotypic treatment studies for childhood precursor-B ALL from April 2000 combinations of antigens that are not present on normal bone 9,22,27–29 to January 2002. Enrollment was based on patients’ NCI risk marrow cells. Both methods are generally recognized groups and the presence of specific molecular abnormalities, as as useful, although they have different advantages and shown in Table 1. All patients were enrolled on the POG 9900 disadvantages. When optimally performed, molecular studies classification study, which required shipment of blood and bone are more sensitive, and may require less material. However, marrow samples to reference laboratories at the University of flow cytometry is less expensive than molecular methods that New Mexico and the Johns Hopkins Medical Institutions. rely on sequencing to detect clone-specific markers, and is Patients with precursor-B-cell ALL, as confirmed by the Johns rapid, with test results available within a few hours of sample Hopkins laboratory, were categorized at the end of induction as receipt. low-, standard-, or high-risk based on modified age and white MRD detected using either method is an important prognostic 42 1,5–8,13,14,26,29–32 blood cell (WBC) criteria and molecular studies performed at factor in the outcome of children with ALL. the University of New Mexico, as described below. These MRD has been studied at multiple time points throughout patients were enrolled on consolidation/continuation treatment treatment. These studies show that although patients are noted protocols 9904, 9905, and 9906, respectively. All patients or to be positive less frequently as treatment progresses, MRD has their parents or guardians gave separate informed consent for the classification and treatment studies. Patients with precursor- Correspondence: MJ Borowitz, Children’s Oncology Group, 440 E. Huntington Dr. Suite 300, PO Box 60012, Arcadia, CA 91066-6012, B ALL enrolled on the classification study 9900 received USA; Fax: +1 626 445 4334 induction therapy of three drugs (vincrinstine, dexamethasone, Received 24 December 2002; accepted 26 March 2003 and L-asparaginase) or four drugs (vincristine, prednisone, Minimal residual disease in childhood ALL MJ Borowitz et al 1567 Table 1 Risk group classification for treatment assignment at day recovery with time in some but not all specimens. Thus, we 29 cannot rule out some level of false negatives among the older specimens. Risk group Definition (Treatment protocol #) Flow cytometry Low-risk group NCI standard risk for induction (POG 9904) (WBC o50 000/ml, Diagnostic samples: Each sample received in the Johns age 1.001–9.999 years) Hopkins reference laboratory that had been classified as No CNS-3* or testicular involvement No adverse translocations precursor–B-cell ALL was studied with a limited panel of (ie E2A-PBX1, BCR/ABL, MLL) antibodies in four-color flow cytometry to confirm the diagnosis, Has one of the following: to identify phenotypes associated with particular translocations, trisomies 4 and 10 by FISH as previously described,27 and to define an abnormal phenotype Tel-AML1 by PCR for purposes of monitoring. In nearly 90% of cases, the two-tube panel CD20ÀFITC/CD10ÀPE/CD45-perCP/CD19ÀAPC and Standard-risk group Not low risk (POG 9905) Not high risk CD34ÀFITC/CD9ÀPE/CD45ÀperCP/CD19ÀAPC was sufficient for this purpose, and defined several abnormal phenotypes High-risk group Does not have trisomies 4 and useful for monitoring for residual leukemia. In the remaining (POG 9906) 10 or Tel-AML1 cases, additional markers including CD38, CD15, CD13,

3 CD66b, CD22, or TdT were added to these combinations to Age (rounded down)/WBC ( Â 10 /ml) within pragmatic high definition, further define an informative phenotype for monitoring. All as follows: antibodies were obtained from Becton Dickinson (San Jose, CA, Age/WBC for day 28 high risk USA) except for CD10 (Dako, Carpenteria, CA, USA) and Age (years) CD66b, CD38, and TdT (Beckman-Coulter/Immunotech, Mia- mi, FL, USA). Cases in which the local institutional diagnosis Boy Girl WBC at diagnosis was not known were initially studied with a larger panel of antibodies to confirm lineage, and cases identified as precursor- 8 12 >80 B ALL were handled as described above. 9 13 >60 10 14 >40 Follow-up samples: Day 8 blood samples were processed 11 15 >20 by a modified stain-then-lyse procedure, usually using the single >12 >16 Any WBC Regardless of other factors if has four-color tube that best discriminated normal and abnormal MLL rearrangement cells. Since most cases had low cell numbers, a procedure was CNS-3a or testicular involvement developed using a buffy-coat-enriched specimen. Serum, buffy coat and the top of the red cell layer were obtained from a Patients >12 months of age with precursor–B-cell ALL [t(9;22) and pelleted cell preparation, and the material resuspended and hypodiploidy o45 chromosomes are not eligible for these studies] processed by our usual stain-then-lyse procedure. When the aCNSÀ3=CSF WBC X5/ml with blasts on cytospin. buffy-coat method was compared in 15 samples to a lyse-then- stain method using ammonium chloride, there was a high correlation (r ¼ 0.99 by linear regression analysis) in the percent of leukemic cells found, with a few samples showing superior recovery with the buffy-coat method. Day 29 marrow samples L-asparaginase, and daunomycin) based on their NCI risk almost always had sufficient cells to process using standard classification at diagnosis. Patients o1 year old, those with methods, but in about 2% of samples, only sufficient material T-cell ALL, Burkitt’s leukemia, Philadelphia chromosome- was available to run a single informative tube. In the remaining positive (Ph+(bcr-abl+)) ALL, or those who did not achieve first cases, at least two and in rare cases three different four-color remission were not eligible for any of these studies. tubes were assayed. Bone marrow was obtained at day 8 to determine morpho- Stained samples were analyzed on a dual-laser FACSCAlibur logic response. Patients were classified as M1 (o5% blasts), M2 flow cytometer (Becton-Dickinson, San Jose, CA, USA). In all (5–24% blasts), and M3 (X25% blasts) by local institutions cases, CD19-APC was used as a gating reagent. Generally using standard morphologic criteria. Additionally, a blood 750 000 cells, with a minimum of 500 000 cells, were run sample at day 8 was obtained for flow cytometric studies of through the flow cytometer for day 29 marrow samples. Only residual disease. At day 29 following induction therapy, bone CD19-positive (CD19+) cells with low right-angle scatter were marrow and blood samples were sent for MRD studies at both saved for further analysis. Debris was subtracted from the the New Mexico and Johns Hopkins reference laboratories. The denominator based on examination of forward scatter vs side MRD studies at New Mexico will be the subject of a separate scatter displays. The total number of B cells, calculated as the report. percentage of CD19+ events divided by the total number of cells In 93% of cases, the specimen was processed within 24 h of run through the cytometer, showed excellent agreement in all obtaining the sample. The remaining 7% of specimens, ranged cases with the percent of CD19+ cells seen in a separate from 3 to greater than 5 days old. We found MRD somewhat less acquisition of 10 000–20 000 ungated events. This latter frequently (18 vs 28% overall) in these older samples. The acquisition was also used to calculate the percent of mono- distribution of cytogenetic abnormalities and of slow early nuclear cells in the sample; granulocytes were excluded on responders by conventional morphology was the same in this displays of CD45 vs right-angle scatter, based on their 7% of patients as in the entire group. Experiments in which intermediate density CD45 and high scatter. In some experi- samples were stored in the laboratory showed a decrement in ments, the combination CD45 and CD71 was used to identify

Leukemia Minimal residual disease in childhood ALL MJ Borowitz et al 1568 nucleated red cells definitively for a more accurate assessment images of 200 interphase nuclei from each specimen were of total mononuclear cells and debris and excellent agreement examined independently by two readers. The presence of a cell was found in the percentages of mononuclear cells with both line characterized by trisomy 4 and/or 10 was established if methods. The percentage of mononuclear cells was used to 46% of nuclei examined contained three hybridization signals correct the percentage of residual leukemic cells in positive with each probe. cases. Leukemic cells were identified based on their abnormal pattern of antigen distribution on multiple simultaneously Statistics methods viewed dual-parameter displays. Normal ‘templates’27 showing the expected pattern of antigen expression in normal B-cell This report is mostly descriptive. Associations involving development were used to screen samples for the presence of MRD and other characteristics were analyzed by Pearson’s w2 abnormal cell clusters in positions outside the normal areas. The or the Kruskal–Wallis test if the covariate had more than two percentage of MRD was determined by dividing the number of (ordinal) categories. Association among MRD, day 8 blood, cells in an abnormal cluster by the total number of cells and day 8 bone marrow were performed by the acquired. Based on prior experiments, it was determined that in Mantel–Haenszel test, for stratified two-by-two tables. In each most cases it was possible to recognize a cluster of as few as 15 case, one variable was stratified for analyzing the other two. events, if these were well defined and distinct from normal All P-values are two-sided. areas. This gives a theoretical sensitivity of 1/50 000 for cases with 750 000 events collected. However, because not every case achieved this ideal, we considered our operational Results sensitivity to be 1/10 000 (generally a minimum of 75 events, which was very easy to see) and most of our results are reported Correlation of MRD and traditional risk factors at this threshold. In about 7% of cases, we could only achieve a sensitivity estimated at 1/1000. Reasons for this included having Overall, 225 of 1016 (22.1%) patients had bone marrows too few cells for analysis or having very large numbers of normal at the end of induction with MRD 40.01%. An additional B-cell precursors and a phenotype that showed partial or 65 patients had detectable MRD at levels below 1/10 000 significant overlap between the leukemic and normal cells. In cells, for a total positivity rate of 28.6%. A total of 129 of 1016 6.6% of cases enrolled on one of the treatment studies, no day (12.7%) patients had MRD levels greater than 0.1%. Among 29 marrow specimens were received, and 1.8% of samples were patients considered positive, the median percent positive was considered to have indeterminate results, usually because no 0.069%. abnormal phenotypes were determined at diagnosis. The MRD correlated significantly with risk group. NCI high-risk number of uninformative cases is lower than that suggested by patients were significantly more likely to be MRD-positive previous reports,2 because in most cases there were very few or (MRD+) than standard-risk patients. Of high-risk patients, 29.8% no normal B-cell precursors seen in day 29 marrows, something (n ¼ 325) were MRD+ 40.01%, compared with 18.5% of that has been previously noted.9 Although we did not record the standard risk (n ¼ 690) (Po0.001 by w2 analysis). At a cutoff of percentage of normal B-cell precursors in all cases, in a subset of 0.1%, there were 22.8% high-risk patients positive, compared 100 consecutive day 29 marrows we found nearly half (48%) to with 8.0% standard risk (Po0.001). The median percentage of have no detectable normal B-cell precursors (ie no events in leukemic cells among patients considered positive was nearly 750 000 cells), with a maximum number of 0.6% normal cells. eight times higher (0.30 vs 0.041%) in NCI high-risk patients Thus, in such cases, even a phenotype that might have fallen than in NCI standard-risk patients. within the normal template boundaries could still easily be recognized as abnormal. Relation between MRD and molecular genetic findings at diagnosis PCR testing and fluorescence in situ hybridization Table 2 shows the relation between MRD levels at the end of Diagnostic samples of bone marrow and peripheral blood induction, and genetic lesions identified at diagnosis. Although received at the University of New Mexico Cancer Center were patients with demonstrable bcr-abl were excluded from the processed by Ficoll–Hypaque density gradient centrifugation, therapeutic studies noted above, 18 patients with Ph+ ALL had followed by standard cell counting and viability studies. material sent for MRD analysis at end of induction; 16 of 18 Aliquots of cells were allocated for DNA index analysis, (89%) were positive, all with high (40.1%) levels of disease. fluorescence in situ hybridization (FISH), and PCR assays. Five of 38 patients (13.2%) with E2A-PBX1 ALL had MRD, Screening for common chromosomal translocations in pediatric and four of five had levels 40.1%. The most surprising ALL, including the t(9;22)/BCR-ABL, t(1;19)/E2A-PBX1, t(12;21)/ findings, however, were that patients with favorable trisomies TEL-AML1, and t(4;11)/MLL-AF4 was performed by reverse- and those with TEL-AML1 translocations had substantially transcription PCR following RNA extraction using standard different levels of MRD detected, even though both are methods. Post-PCR detection of specific RT-PCR products was considered good-risk patients. At a threshold of 0.01%, 20.3% confirmed by chemiluminescent Southern blot hybridization of patients with trisomies 4 and 10 were MRD+, compared with junctional-region oligonucleotide probes. All pediatric with only 8.2% of TEL-AML1 patients; nearly 10% of good samples with DNA indexes 41.0 had centromeric probe FISH trisomy patients had high levels of MRD (40.1%) compared testing performed for trisomies of chromosomes 4 and 10 to with 2.4% of those with TEL-AML1. It should also be noted that identify ‘good-risk’ hyperdiploid patients. Cells were cohybri- there was a similar relation in the morphologic response to dized with centromere enumeration probes for chromosomes 4 therapy between the TEL-AML1 and the good trisomy patients; (Spectrum Oranges) and 10 (Spectrum Greens) from Vysis, Inc. 24% of trisomies 4 and 10, but only 10% of TEL patients, had (Downers Grove, IL, USA). After hybridization, the fluorescent M3 marrows at day 8.

Leukemia Minimal residual disease in childhood ALL MJ Borowitz et al 1569 Table 2 Correlation of day 29 MRD and genetic abnormalities at Table 3 Relation of ﬂow cytometric detection of residual leukemia diagnosis in day 8 blood specimens with day 8 marrow morphology, and day 29 MRD Abnormality MRD >0.1% MRD >0.01% Fraction of patients MRD+at day 29 BCR-ABL 16/18 (89%) 16/18 (89%) E2A-PBX1 4/38 (10.5%) 5/38 (13.2%) Day 8 morphology Day 8 blood negative Day 8 blood positive TEL-AML1a 5/208 (2.4%) 17/208 (8.2%) Trisomies 4 and 10a 20/207 (9.7%) 42/207 (20.3%) M1 4/258 (1.6%) 25/255 (9.8%) aMRD frequencies are signiﬁcantly different between TEL-AML1 M2/M3 2/51 (3.9%) 70/260 (27%) patients and favorable trisomy patients (P=0.001, Kruskall-Wallis test). Totals 6/309 (1.9%) 96/515 (18.6%)

determined whether day 8 marrow morphology or day 8 blood flow cytometry was a better predictor of bone marrow MRD at day 29. All three variables were satisfactorily measured in 824 patients. The results are shown in Table 3. Day 8 marrow morphology and day 8 blood flow cytometry were independent factors predictive of day 29 bone marrow MRD. The associations among all three variables are significant, and stratification on any one shows that the other two are still significantly correlated (Po.001, Mantel–Haenszel test). Patients who had no detectable blood blasts by flow cytometry at day 8 were rarely MRD+ at day 29, even if they were M2 or M3 by morphology. However, patients positive by flow cytometry in the day 8 blood samples were nearly three times more likely to Figure 1 Relationship of MRD and day 8 marrow morphology. be MRD+ if they had 45% morphologically detectable blasts in The percentage of patients with MRD positivity at end of induction at the day 8 marrows. This suggests that while day 8 blood flow both the 0.1 and 0.01% levels is plotted against marrow blast content cytometry measurements cannot replace marrow assessment of based on morphologic examination of a day 8 marrow sample. At both levels MRD and blast content are positively associated. early response, they might help to indicate which patients could benefit from marrow examination.

Correlation of end-of-induction marrow MRD and day 8 bone marrow morphology Discussion

Morphologic data on day 8 bone marrow samples were Many studies over the past several years have suggested that the available for 936 patients with MRD data. Day 8 bone marrow presence of MRD early in therapy for childhood ALL is a strong morphology showed that 577 patients had M1 marrows, 157 adverse prognostic factor.1–11,13–15 Although some of these patients had M2, and 202 had M3. As shown in Figure 1, there studies demonstrated that MRD maintained prognostic signifi- was significant correlation between residual disease assessed by cance in Cox-model multivariate analyses, these studies bone marrow morphology and day 29 MRD either at 0.1 or generally have not had enough patients to understand the 0.01% (Po0.001 for both by Kruskal–Wallis analysis). How- relation between MRD and other risk factors, or to determine if ever, the correlation was not perfect. Of rapid early responders there was any interaction among variables. That is, it is not clear with day 8 M1 marrows, 12% were MRD+ 40.01%, nearly half if the prognostic significance of finding MRD is the same in all (5.5% overall) with high (40.1%) MRD. Similarly, only about patient groups. In the current study, we investigated bone half of patients with day 8 M3 marrows had detectable MRD at marrow MRD levels at the end of induction in more than 1000 the end of induction. children with precursor-B ALL, and correlated this to other known risk factors. This study is currently less than 3 years old, so we do not yet have outcome data. However, this preliminary Assessment of day 8 blood samples by flow cytometry report shows a strong correlation of MRD with other known poor-risk factors. Owing to the known importance of early response to therapy, Overall, we detected bone marrow MRD by four-color flow flow cytometric assessment also was performed at day 8 cytometry at a level of 0.01% in approximately 22% of patients following initiation of therapy. At that time, most marrows are at the end of induction, with 12.7% having more than 0.1%. hypocellular, and it was reasoned that flow cytometric assess- However, at the higher cutoff levels, nearly three times as many ments of bone marrow would be inaccurate because of NCI high-risk patients were MRD+ compared with NCI hemodilution, especially since the first aspirate of this marrow standard-risk ones, suggesting that poor clearance of leukemic was invariably used for morphology. Accordingly, we used cells may explain high-risk characteristics. This interpretation is blood specimens at day 8, in the hope that this would provide a consistent with laboratory studies showing that MRD was more accurate measurement. Overall, 553 of 909 (60.8%) correlated with poor in vitro prednisolone response.43 Similarly, patients had 40.01% leukemic cells detected in blood at day 8. patients with slow early response, as assessed by examination of The median value among positive patients was 0.37%; 44% of marrow morphology at day 8, also were far more likely to be patients had 40.1% and 19.7% of patients 41%. We next MRD+ by flow cytometry at end of induction compared with

Leukemia Minimal residual disease in childhood ALL MJ Borowitz et al 1570 rapid early responders. As it has been shown that slow early comparable or lower.12 Confounding interpretation are varying responders can be rescued following intensification of ther- proportions of patients in different risk groups, especially T-cell apy,44 there is cause for optimism that many patients with MRD ALL, which were excluded from our series but are present in at day 29 can be rescued by such intensification. The current others; patients with T-ALL have been shown to have a higher study is designed to address this issue. incidence of MRD positivity than precursor-B ALL patients.50 In addition to the widely known effects of risk group and early Nevertheless, we recognize that it is possible that in our study, marrow response, genetic abnormalities are known to have a in which more than 100 institutions obtain bone marrow and strong effect on prognosis in precursor–B-cell ALL. This study ship samples, there could be variability of technique in evaluated the rate of end-of-induction MRD in a large number of obtaining bone marrow. This is further magnified in our studies, patients in which PCR or FISH was used to identify the most in which up to 8 ml of marrow are obtained, for flow and important genetic abnormalities in ALL. Not surprisingly, most molecular MRD studies as well as cell banking. Thus, it is patients with Ph+ ALL were MRD+ at high levels, in agreement possible that there may be blood contamination in some of our with other works that noted that Ph+ patients are virtually specimens. However, although this could potentially produce always MRD+ at the end of induction.1 false negatives, a positive finding would still be useful to identify The presence of the TEL-AML1 translocation and hyperdi- patients who might benefit from enhancement of therapy. Study ploidy involving trisomies 4 and 10 have both been associated designs to identify patients to receive less therapy on the basis of with good outcome.37,38,45 In this regard, finding a very low rate lack of MRD should be designed in such a way that the ‘first of MRD positivity among TEL+ patients is not surprising. Only pull’ marrow is used for MRD assessment. 8% of patients had 40.01% MRD positivity at the end of Owing to the potential problem of blood contamination of induction, and less than 3% had 40.1%. Others have noted a marrow, it would be desirable if MRD studies could be similar trend, with smaller numbers.1 Patients with TEL performed on the blood. However, definitive data on whether translocations also have been shown to be rapid early blood positivity can predict outcome do not yet exist. We found responders with conventional therapy.46 Although it is possible that only a small percentage of patients with marrow positivity that some of the patients with this translocation who relapse are positive in the blood at day 29, and this is generally, might correspond to the few patients who are MRD+, this seems although not invariably, at a much lower level, thus requiring unlikely, especially since many relapses appear to occur late in higher sensitivity methods to detect positive patients. Others TEL-AML1 ALL.47–49 Thus, the mechanism of relapse in this have found similarly that blood MRD levels do not match those genetically defined subgroup may be different and not due to of marrow in precursor-B-cell ALL.51–53 In a small series of persistence of disease at the end of induction therapy. patients those with positive findings in blood at end induction Additional studies using DNA microarray technology are in had a very high risk of relapse.53 Our data need further progress in the hopes of identifying novel markers of relapse in maturation to correlate blood end-of-induction results and this patient subgroup. outcome. On the other hand, detection of very rapid clearance The situation with favorable trisomies is quite different. These of MRD from the blood might be useful to anticipate leukemia patients have long-term event-free survival (EFS) of 90%, with regression in marrow. For this reasons, we also investigated day nearly 95% EFS at 3 years38 (Sutcliffe, submitted for publica- 8 blood MRD by flow cytometry. Far too many patients are tion). In spite of an outcome that is as good or better than the positive in the blood at day 8 by flow cytometry for it to be favorable outcome of the TEL+ patients, these patients have a possible to use simple persistence of leukemic blasts as an two- to three-fold higher incidence of MRD positivity at either indicator for early intervention, although some higher value the 0.1 or 0.01% levels, with more than 20% of patients cutoff might turn out to be more predictive of relapse. We also positive. Pending longer follow-up, we do not know whether found interesting correlations between day 8 blood flow favorable trisomy patients with high MRD levels at the end of cytometry and morphologic measures of early marrow response, induction have a higher risk of relapse than those without. It is particularly as they relate to finding end-of-induction marrow also possible that the kinetics of response in this subgroup is MRD. Specifically, patients who are negative by flow cytometry different, and that a later time point might better help to identify at day 8 are almost never MRD+ at day 29, even if they are slow patients at risk for relapse. However, it is clear from results of early responders. It is thus possible that day 8 blood flow could previous trials that early intensification for all patients with be used to identify patients who might benefit from, or might not favorable trisomies, who are MRD+ at the end of induction, need, a marrow examination. Our future plans include day 15 would be overtreatment for a large portion of such individuals. marrow examinations in some patients to help define early Our overall incidence of end-of-induction MRD is lower than marrow response. Patients who are negative by flow at day 8 that seen in the largest published series using molecular may not need this marrow examination. methodology.14 There are several reasons that this might be so. First, the assumptions used in quantitation are very different between methods. What is called 0.01% by flow cytometry may Acknowledgements not be the same as 0.01% by PCR. More significantly, we routinely exclude apoptotic cells from analysis based on light- This work was supported by NIH grants R01 CA86011 and U10 scatter gating, while DNA-based methods will include the DNA 29139. of those cells. We, of course, do not know which is ‘correct,’ but it is not surprising that there is a difference. References The largest flow-cytometry-based series also shows a slightly higher end-of-induction MRD rate than we see here,1 although 1 Coustan-Smith E, Sancho J, Hancock ML, Boyett JM, Behm FG, both the therapy and timing of end-of-induction sampling in that Raimondi SC et al. Clinical importance of minimal residual disease protocol are very different from what we have employed. A in childhood acute lymphoblastic leukemia. Blood 2000; 96: 2691–2696. more recent, smaller series, using three- or four-drug induction 2 Campana D, Neale GA, Coustan-Smith E, Pui CH. Detection of 9 comparable to what we used also showed an MRD+ rate that minimal residual disease in acute lymphoblastic leukemia: the St was much higher, while another recent small series was Jude experience. Leukemia 2001; 15: 278–279.

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