Combined analysis of minimal residual disease at two time points and its value for risk stratification in childhood B-lineage acute lymphoblastic leukemia

Leukemia Research 34 (2010) 1314–1319

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Combined analysis of minimal residual disease at two time points and its value for risk stratification in childhood B-lineage acute lymphoblastic leukemia Lei Cui a , Zhigang Li a,∗ , Minyuan Wu a , Weijing Li a , Chao Gao a , Guoren Deng b a b

Hematological Center, Beijing Children’s Hospital Affiliated to Capital Medical University, 56 Nanlishi Road, Beijing 100045, China School of Medicine, University of California, San Francisco, CA, USA

a r t i c l e

i n f o

Article history: Received 16 November 2009 Received in revised form 16 November 2009 Accepted 30 November 2009 Available online 19 January 2010 Keywords: Minimal residual disease Childhood acute lymphoblastic leukemia Immunoglobulin Gene rearrangement Real-time quantitative PCR

a b s t r a c t The study was aimed to explore the value of minimal residual disease (MRD) for risk stratification in childhood precursor-B-acute lymphoblastic leukemia. MRD was monitored at two time points (TP1, after induction and TP2, before consolidation therapy) by quantitative detection of monoclonal immunoglobulin heavy chain gene rearrangements. This study stratified 105 patients into three MRD risk groups: standard-risk, MRD < 10−4 at both TP1 and TP2; high-risk, TP1 ≥ 10−2 or TP2 ≥ 10−3 ; and others were classified as intermediate-risk. We incorporated this MRD risk information to refine risk stratification among these patients and developed a new classification system that predicted the treatment outcomes more successfully than did the traditional risk classification criteria. © 2009 Elsevier Ltd. All rights reserved.

1. Introduction Over the past decade, progress in treatment protocols has resulted in a great improvement in the overall prognosis of childhood acute lymphoblastic leukemia (ALL) [1,2]. However, up to 20% of patients relapsed due to insufficient therapy, while some patients were over-treated and consequently suffered from treatmentrelated toxicity [3–5]. Precise risk-directed treatment may improve survival rates through identification of patients with heterogeneous early responses to therapy. Assessment of minimal residual disease (MRD) has been shown to be a more powerful technique for assessment of treatment response than traditional morphologic monitoring. Therefore, it has been used to stratify risk and guide therapy in multiple childhood ALL studies [6–8]. The time points for MRD monitoring and the cut-off levels for risk classification vary depending on the treatment protocol. The Berlin-Frankfurt-Munster (BFM) group adopts the most stringent MRD assessment of two time points (day 33 after induction and week 12 before consolidation) [6,9], whereas the Dana-Farber Cancer Institute (DFCI) ALL Consortium uses day 30 at the end of induction as a single time point for MRD detection [8]. The methods used for quantitative MRD detection differ among protocols, with some studies using polymerase chain reaction (PCR) techniques and

∗ Corresponding author. Tel.: +86 010 68028401x2622; fax: +86 010 68055864. E-mail address: [email protected] (Z. Li). 0145-2126/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2009.11.031

others using flow cytometry [10–16]. Detection of clonal rearrangements of immunoglobulin (Ig) and T-cell receptor (TCR) genes using real-time quantitative PCR (RQ-PCR) is the most widely employed strategy for MRD monitoring in ALL [17–19]. In this study, we monitored MRD at two time points: day 33 after induction and week 12 before consolidation. MRD was measured using RQ-PCR-based quantification of leukemic IgH gene rearrangements. The patients were stratified into three MRD risk groups according to their MRD status at two time points. Subsequently, we incorporated information on MRD and conventional risk factors in order to refine risk stratification. We identified a significantly different risk stratification system that contrasts with those based on clinical risk groups. This is the first time the value of MRD for risk stratification was explored in Chinese children with precursor-Bacute lymphoblastic leukemia (precursor-B-ALL). 2. Materials and methods 2.1. Patients and treatment From 1 April 2005 to 31 December 2007, 270 children aged between 1 and 16 years with newly diagnosed precursor-B-ALL enrolled in the therapy study ‘Beijing Children’s Hospital BCH-ALL 2003’ at our institution. The patients were stratified into standard-risk, intermediate-risk and high-risk treatment groups (SRG, IRG and HRG) according to age, white blood cell (WBC) count, immunophenotype, cytogenetic and molecular aberrations, prednisone response and morphological remission after 33 days of induction therapy (based on BFM risk criteria). The stratification criteria and treatment protocol are described in Fig. 1. The BCH-ALL 2003 protocol was approved by Beijing Children’s Hospital Institutional Ethics Committees, including the informed consents signed by guardians of each patient.

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Fig. 1. Induction, early intensification and consolidation treatment protocol used in BCH-ALL 2003. MRD analysis was performed after induction (TP1) and before consolidation therapy (TP2) (TP2 in HRG is prior to the second cycle of HR-1 block). Induction treatment (VDLP) included a prednisone backbone, vincristine, daunorubicin and lasparaginase. Daunorubicin was given as 2 doses in SRG and as 4 doses in MRG and HRG. Early intensification treatment (CAM) in SR and MR group was based on 6mercaptopurine with pulses of cyclophosphamide and cytarabine. High-dose methotrexate (HD-MTX) was used as a consolidation block in the SR and MR groups. BFM 6 HR blocks after induction treatment were adopted for the HRG group.

In this study, 105 patients with precursor-B-ALL were included in the MRD analysis. Each patient fulfilled the following criteria: (1) sufficient DNA samples harvested at diagnosis, day 33 and week 12 were available for analysis; (2) a monoclonal IgH gene rearrangement was identified at diagnosis; (3) an IgH target was present with a quantitative range of at least 10−4 , to allow for detection by RQ-PCR. Two patients were excluded from MRD analysis because of induction failure or induction death. No significant differences were observed between these included (n = 105) and not included (n = 165) in the MRD analysis for treatment group (P = 0.312), age group (P = 0.174), sex (P = 0.444) and WBC count group (P = 0.177). The 3-year overall survival and relapse-free survival in the group with successful MRD monitoring were 89 ± 4% and 84 ± 4%, compared with the group of patients not included in the MRD analysis were 91 ± 2% (P = 0.871) and 88 ± 3% (P = 0.960).

2.2. Cell samples and DNA isolation Bone marrow (BM) samples were obtained at diagnosis, at day 33 (TP1) and during week 12 (TP2) of therapy. BM mononuclear cells (MNC) were isolated by Ficoll gradient centrifugation (MDPACIFIC, Tianjin, China, density: 1.077 g/mL) and stored at −70 ◦ C for DNA extraction. Normal peripheral blood cells were obtained from healthy volunteers; MNC from ten donors were pooled. Genomic DNA was extracted from MNC using the Blood DNA Kit (U-gene, Anhui, China).

2.3. Identification of PCR targets and design of ASO primers We used patient-specific IgH gene rearrangements as RQ-PCR targets for quantitative assessment of MRD in precursor-B-ALL. Complete IgH gene rearrangements were amplified in the patient’s diagnostic samples by multiplex PCR, using the BIOMED-2 primer sets for IgH tubeA [20]. If only one band of expected size was identified, the PCR products were sequenced using an ABI PRISM 3730 Automated Sequencer (Shanghai Sangon Biological Engineering Technology & Service Co., Ltd.). Junctional region sequences were analyzed using the IMGT (http://www.imgt.cines.fr) or IgBlast (www.ncbi.nlm.nih.gov/igblast/) database.

We applied an RQ-PCR approach for IgH gene rearrangements using one of the JH germline TaqMan probes and primers in combination with an allele-specific oligonucleotide (ASO) primer complementary to the junctional region. The germline JH TaqMan probes and primers have been described previously [21]. 2.4. MRD RQ-PCR analysis and quantitation of MRD levels RQ-PCR analysis was performed using the ABI PRISM 7000 Sequence Detection System (PE Biosystems). Reaction mixtures contained 1× QPCR ROX Mix (AB gene, England), 3.125 pmol upper and lower primers, 1.25 pmol probe, 0.3% bovine serum albumin (BSA) and 600 ng DNA. The reaction was carried out at 95 ◦ C for 15 min, followed by 50 cycles of 15 s at 95 ◦ C and 1 min at 54–66 ◦ C [22]. To determine the efficiency of amplification and sensitivity of MRD PCR targets, diagnostic DNA was serially diluted tenfold in normal MNC DNA (from 10−1 to 10−5 ) and used in duplicate for RQ-PCR amplification to generate a standard curve, normal control MNC DNA was amplified in triplicate. In patients with a quantitative range of MRD target ≤10−4 , the follow-up samples at the two time points were subjected to RQ-PCR analysis in triplicate. To check the quantity and quality of the DNA in the assay, N-ras gene was used as a control gene for RQ-PCR analysis [23]. The MRD level is expressed as the normalized IgH value, i.e., IgH gene number divided by N-ras gene number. The RQ-PCR settings were chosen and interpretation of real-time quantitative data was performed following the guidelines of the European Study Group on MRD detection in ALL (ESG-MRD-ALL) [24]. 2.5. Statistical analysis Relapse-free survival (RFS) was defined as the time from complete remission to the date of relapse, censored at date of last time of contact or remission death. The probability of RFS for each patient was estimated using the Kaplan–Meier method and univariate associations between risk groups were compared by log-rank tests. Differences in the distribution of clinicobiological features by MRD risk group were compared using the exact chi-square test and multivariate analysis of the pre-

Table 1 Distribution of MRD values at two time points and relapse-free survival (RFS) outcome. MRD level

Negative Positive, <10−4 ≥10−4 and <10−3 ≥10−3 and <10−2 ≥10−2

Time point 1

Time point 2

Samples

Relapsed

RFS

SE

Samples

Relapsed

RFS

SE

43 21 20 14 7

2 1 2 5 3

0.93 0.95 0.90 0.61 0.25

0.05 0.05 0.07 0.14 0.22

80 14 5 1 5

3 4 2 1 3

0.95 0.71 0.33 0

0.03 0.12 0.28

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dictive value of MRD was performed by Cox-regression analysis. The disparity in distribution between MRD-directed risk stratification and clinical classification was evaluated using the exact chi-square test. P-values <0.05 were considered significant.

3. Results 3.1. MRD PCR target availability and sensitivity in precursor-B-ALL patients PCR target identification was performed on 270 precursor-BALL patients for whom sufficient high-quality DNA was available at diagnosis. Of these initial diagnostic samples, clonal complete VH –JH gene rearrangements were detected in 230 patients (85.2%) and monoclonal rearrangements were identified in 134 (49.6%). Of the 134 children with monoclonal rearrangements, 12 were excluded from further analysis due to lack of day 33 or week 12 BM samples (10), induction failure (1) or induction death (1). The remaining 122 cases were analyzed for RQ-PCR sensitivity. The required quantitative range of 10−4 to 10−5 was reached in 105 patients (86.1%); therefore quantitative detection of MRD was performed in these patients. The correlation coefficients of all standard curves were above 0.98. The mean slope of the standard curves was −3.35 ± 0.18 and the mean intercept was 21.25 ± 1.15. The lowest CT values of negative control samples were above 40 cycles, and they were ≥3.0 CT higher than the highest CT value of the quantitative rage. Each DNA sample was quantified the number of N-ras copies for control quantity and quality, and the number of amplified genes in each sample varied by less than twofold. 3.2. Stability of PCR targets at relapse A total of 13 patients relapsed, including 12 relapses in BM. One patient had an isolated extramedullary relapse in the central nervous system. In patients with BM relapse, BM samples at clinical relapse were analyzed for PCR target stability. The PCR targets remained stable in 10 patients (83.3%), while two targets at relapse were different from targets at diagnosis, due to continuing rearrangements or to clonal selection. 3.3. Prognostic value of MRD and MRD-based stratification A total of 105 children with precursor-B-ALL were evaluable by MRD analysis. Thirteen patients relapsed, the median followup was 30 months and the estimated 3-year RFS rate was 84 ± 4%. The percentage of patients who were MRD-positive decreased from 59% (62 of 105) at TP1 to 24% (25 of 105) at TP2. According to their evaluated MRDs at TP1 and TP2, patients were stratified into five groups: MRD-negative, MRD-positive with <10−4 , ≥10−4 and <10−3 , ≥10−3 and <10−2 , or ≥10−2 . Three-year RFS in these groups of patients was assessed (Table 1). We further evaluated the significance of different MRD levels (Table 2). We found that an MRD < 10−4 at TP1 and MRD-negative status at TP2 predicted excellent outcome, while an MRD ≥ 10−4 at TP1 Table 2 Comparison of relapse-free survival in patients grouped by MRD level. Time point

MRD cut-off

Log-rank test

Time point 1

Negative/positive <10−4 /≥10−4 <10−3 /≥10−3 <10−2 /≥10−2

P = 0.063 P = 0.003* P < 0.001* P = 0.012*

3.780 5.612 7.360 4.526

Negative/positive <10−4 /≥10−4 <10−3 /≥10−3

P < 0.001* P < 0.001* P < 0.001*

12.793 9.954 15.838

Time point 2

*

Significant (P < 0.05).

Hazard ratio

Fig. 2. Comparison of the probabilities of relapse-free survival in three MRD risk groups by log-rank tests. A significant trend (P < 0.001) was noted. Patients with MRD < 10−4 at both TP1 and TP2 were classified as the standard-risk group (MRDSR); TP1 ≥ 10−2 or TP2 ≥ 10−3 as the high-risk group (MRD-HR); others as the intermediate-risk group (MRD-IR).

(P = 0.003) and MRD-positive status at TP2 (P < 0.001) was significantly associated with relapse. Because it was difficult to quantify MRD levels below 10−4 in most patients, we used 10−4 as the cut-off point at both TP1 and TP2 for identification of patients with a low risk of relapse. We also determined that patients with MRD ≥ 10−2 at TP1 or MRD ≥ 10−3 at TP2 had the worst outcomes (3-year RFS of 25% and 0%, respectively); therefore, 10−2 at TP1 and 10−3 at TP2 were used to identify patients with a high-risk of relapse. We subsequently divided the 105 patients into three MRD risk groups based on their MRD levels at TP1 and TP2 (Fig. 2): a standardrisk group (MRD-SR), with MRD < 10−4 at both TP1 and TP2; a high-risk group (MRD-HR), with TP1 ≥ 10−2 or TP2 ≥ 10−3 ; and an intermediate-risk group (MRD-IR) including the rest of the patients. Table 3 MRD level according to clinicobiological features. Variables

P value*

MRD group MRD-SR (%)

MRD-IR (%)

MRD-HR (%)

Total number

64

30

11

Treatment group SRG IRG HRG

24(61.5) 38(71.7) 2(15.4)

12(30.8) 10(18.9) 8(61.5)

3(7.7) 5(9.4) 3(23.1)

0.003

Age (y) <1 1–9 ≥10

2(100) 52(65) 10(43.5)

0 22(27.5) 8(34.8)

0 6(7.5) 5(21.7)

0.154

Sex Male Female

34(56.7) 30(66.7)

19(31.7) 11(24.4)

7(11.7) 4(8.9)

0.642

WBC (109 /L) <20 ≥20

43(63.2) 21(56.8)

17(25) 13(35.1)

8(11.8) 3(8.1)

0.588

BCR-ABL Absent Present

64(63.4) 0

27(26.7) 3(75.0)

10(9.9) 1(25.0)

0.023

TEL-AML1 Absent Present

50(58.1) 14(73.7)

25(29.1) 5(26.3)

11(12.8) 0

0.246

E2A-PBX1 Absent Present

59(59.0) 5(100)

30(30.0) 0

11(11.0) 0

0.288

Prednisone response Good 62(63.9) Poor 2(25.0)

25(25.8) 5(62.5)

10(10.3) 1(12.5)

0.067

WBC, white blood cell count. * By exact chi-square test for independence.

L. Cui et al. / Leukemia Research 34 (2010) 1314–1319 Table 4 Comparison of stratification by conventional risk criteria with stratification by MRDincorporation assessment. Incorporated MRD stratification SR Clinical stratification SRG 24(0) IRG 0 HRG 0 Total

24(0)

Total

IR

HR

12(0) 48(3) 0

3(1) 5(4) 13(5)

60(3)

21(10)

39(1) 53(7) 13(5) 105(13)

Of our study population, 64 (61%) of the patients were classified as MRD-SR, 30 (29%) as MRD-IR and 11 (10%) as MRD-HR. The 3-year RFS rates were 94 ± 3%, 86 ± 6% and 18 ± 16%, respectively (P < 0.001). 3.4. Relationship between MRD and clinicobiological features We tested the correlation between MRD levels and traditional risk factors. The MRD risk classification significantly correlated with clinical risk stratification (treatment group), although MRD risk was not significantly related to age, sex, WBC count or prednisone response (Table 3). The HRG patients (i.e., patients with BCR-ABL, poor prednisone response, or lacking cytomorphologically complete remission on day 33) were notably more likely to have higher degrees of MRD than the other two treatment groups. In addition, 23% of HRG patients were MRD-HR, compared with 8% of SRG and 9% of IRG patients (P = 0.003). A relationship was observed between MRD levels and molecular genetic findings at diagnosis: all 4 patients with the BCR-ABL fusion gene had high MRD levels (>10−3 ) at TP1 and none of these patients were classified MRD-SR. This trend contrasts with the low MRD values in most (14 of 19) cases with TEL-AML1 and all 5 cases with E2A-PBX1; all of these patients were classified as MRD-SR. Multivariable analysis of relapse rates included MRD stratification, treatment group, age, WBC count and detection of the fusion gene BCR-ABL. MRD remained a significant independent prognostic factor (P = 0.008). Children in the MRD-HR group had 12 (95% CI, 2.5–12.5)-fold greater risk of relapse than those in the MRD-SR group (P = 0.002). 3.5. Re-stratification according to MRD level As shown in Table 4, we incorporated the information obtained on MRD at two time points to refine risk stratification in these 105 patients. Of clinical SRG patients, 31% (12/39) were brought into the

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IR group and 8% (3/39) were brought into the HR group. 9% (5/53) of clinical IRG patients were shifted into the HR group. All clinical HRG patients remained in the HR group, regardless of their MRD levels. With the combined use of MRD assessment and conventional risk criteria, 23% (24/105) of patients were classified as SR, 20% (21/105) as HR and the others (57%, 60/105) as IR according to the new risk group classification. The new classification showed significant disparity with the distribution of clinically assigned risk groups (the percentages in clinical SRG, HRG and IRG were 37%, 13% and 50%, respectively) (P < 0.001). The 3-year RFS for SR, IR and HR according to the new risk group stratification were 100%, 93 ± 4% and 39 ± 13%, respectively, as compared to 97 ± 2%, 82 ± 6% and 54 ± 16% according to the original clinical stratification (Fig. 3). 4. Discussion Using patient-specific IgH gene rearrangements as RQ-PCR targets in 105 children with precursor-B-ALL, we quantitatively detected MRD levels after induction and before consolidation treatment and re-stratified these patients according to their MRD levels. We used tubeA primer sets corresponding to VH FR1 regions to amplify VH –JH rearrangements in childhood precursor-B-ALL, referring to standardized multiplex PCR protocols for detection of Ig/TCR gene rearrangements developed by European BIOMED2 Concerted Action [20]. Previous studies have demonstrated that >95% of childhood precursor-B-ALLs exhibit clonal IgH gene rearrangements and that the vast majority of IgH gene rearrangements represent complete VH –(DH )–JH joining [19,25–27]. We thus chose IgH as a target for MRD monitoring. The overall detection rate in this study was 87.4%. Oligoclonality at the IgH locus can lead to difficulty in choosing one locus for MRD monitoring [19], and the secondary or ongoing gene rearrangements that occur during the time period between diagnosis and relapse can result in false-negative MRD results [28,29]. Fortunately, monoclonal MRD PCR targets in childhood precursor-B-ALL are highly stable. Design of primers around the relatively stable DH –JH region helped to avoid the loss of MRD targets [19,30,31]. Therefore, the sensitivities in the MRD detection targeting monoclonal IgH rearrangements were at least 10−4 , and the IgH rearrangements at relapse remained stable in most (83%) patients. As many reports have shown, IgH rearrangements represent the most sensitive group of Ig/TCR targets. We tested 125 IgH gene rearrangements by RQ-PCR. Quantitative ranges of at least 10−4 were achieved in 86.1% of patients. However, amplification failed in the remaining 13.9% of patients because of low sensitivity or high background, indicating that not all evaluated IgH junctional regions

Fig. 3. Comparison of relapse-free survival (RFS) in standard-risk group (SR), intermediate-risk group (IR) and high-risk group (HR) identified by clinical stratification (A) and by MRD-incorporated stratification (B).

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were suitable for ASO design in MRD detection; and other Ig/TCR targets should be tested in these patients. Previous studies have suggested that the MRD level early in therapy is a powerful prognostic factor for childhood ALL [32,33]. These studies unequivocally demonstrated that the MRD value after induction (TP1) and before consolidation treatment (TP2) could be used to predict relapse. Since analysis of MRD at one time point is insufficient for predicting the outcomes [9,15], we stratified patients into three MRD risk groups with combined use of MRD at TP1 and TP2. Among our study population, 61% were classified as MRD-SR (MRD < 10−4 at both TP1 and TP2), 10% as MRD-HR (TP1 ≥ 10−2 or TP2 ≥ 10−3 ) and 29% as MRD-IR. A significant log trend of 3-year RFS in the patients stratified by MRD detection was noted. The MRD-HR group displayed a significantly worse average outcome than the MRD-SR and MRD-IR groups. While we confirmed MRD as a significant independent prognostic factor using multivariate analysis [32], examination of the relationship between MRD levels and conventional risk factors revealed a strong correlation between MRD and other known poorrisk factors. The percentage of patients at the MRD-HR risk level was markedly higher in HRG than in SRG and MRG, suggesting that poor clearance of leukemic cells may lead to relapse and poor outcomes. The clinical significance of MRD levels may also differ in cytogenetic subgroups of ALL. MRD rates were particularly high in BCR-ABL-positive patients consistent with other reports about the inferior outcomes in such patients [15,34]. In contrast, MRD rates were notably low in most TEL-AML1-postive patients, who were in continuous complete remission. Nevertheless, several TEL-AML1positive patients were classified as MRD-IR. One of them relapsed, indicating that patients with TEL-AML1 translocation display heterogeneous responses to treatment. All of the patients with an E2A-PBX1 translocation were at low MRD risk; E2A-PBX1-positive patients have been shown to be rapid early responders to current cytotoxic therapy protocols and generally had greatly improved outcomes. Serial monitoring of the degrees of MRD was particularly powerful for assessment of treatment response; consequently, it should be incorporated to improve therapy classification. In this study, after re-stratification according to MRD level, 12 cases and 3 cases of clinical SRG patients were shifted into the IR and HR groups, respectively, while 5 cases of clinical IRG patients were transferred into the HR group. These patients showed poor response to current therapy and might be expected to have particularly bad outcomes. They were strongly advised to consider more intensive therapy or stem cell transplantation. SRG patients with MRD-SR, as well as IRG patients with MRD-SR or MRD-IR, remained in their original risk categories, demonstrating good response to current therapy. IRG patients with MRD-SR might benefit from treatment reduction due to decreased treatment-related toxicity. The HRG patients had higher degrees of MRD than the other two treatment groups, indicating a lower clearance rate of leukemic cells and a reduced response to chemotherapy in these patients. Based on these findings, the patients with high-risk characteristics were kept in the HR group despite their MRD risk classifications. The information on MRD levels enabled more precise identification of patients at low and high-risks of relapse: the 3-year RFS for SR and IR were elevated to 100% and 93%, respectively, and that for HR was decreased to 39% due to identification of patients with poor response to chemotherapy, compared to 97% (SRG), 82% (IRG) and 54% (HRG), respectively, in the original clinical stratification. Since MRD has been shown to be highly informative regarding risk modification, it is being applied to improve therapy stratification in several ALL protocols, including in AEIOP/BFM2000, DFCI95-01 and St. Jude XV studies. A Multi-center Chinese Childhood Leukemia Group (CCLG) 2008 study used MRD levels detected by both flow cytometry and RQ-PCR MRD methods on day 33 and

at week 12 as a guide for treatment intensification. This prospective CCLG study adopts a similar MRD risk stratification to that used in the present study. These studies should confirm whether MRDbased individualized therapy improves the outcome of childhood ALL. In summary, this study demonstrates that MRD detection by RQPCR, performed at two time points, is a powerful prognostic tool. The presented findings can be used in the design of novel childhood ALL protocols with MRD-based stratification in China. Conflict of interest The authors declare that they have no conflicts of interest. Acknowledgements This work was supported in part by a grant-in-aid from Beijing Municipal Science & Technology Project (No. D0905001040431), a project of the National Key Technologies Research & Development Program of the 11th 5-Year Plan (No. 2007BAI04B03) and Beijing Novel Program (No. 2005B06). We gratefully thank Ningzhi Xu and Shujun Chen for their excellent technical assistance and final approval. We also thank for all staff of hematology center of Beijing Children’s Hospital affiliated to Capital Medical University for their help in sampling. Contributions. L.C. performed the experiments and drafted the paper. Z.G.L. contibuted to the designs, analysis and interpretation of article, and provided final approval. M.Y.W. provided administrative support, critical revision and final approval of article. W.J.L. and C.G. participated in the experiments in identification of PCR targets and assisted in collection of data. G.R.D. provided excellent technical support and gave critical revision of the manuscript. References [1] Pui CH, Campana D, Evans WE. Childhood acute lymphoblastic leukaemia – current status and future perspectives. Lancet Oncol 2001;2:597–607. [2] Pui CH, Robison LL, Look AT. Acute lymphoblastic leukaemia. Lancet 2008;371:1030–43. [3] Schrappe M, Camitta B, Pui CH, et al. Long-term results of large prospective trials in childhood acute lymphoblastic leukemia. Leukemia 2000;14:2193–4. [4] Gaynon PS, Trigg ME, Heerema NA, et al. Children’s cancer group trials in childhood acute lymphoblastic leukemia: 1983–1995. Leukemia 2000;14:2223–33. [5] Izraeli S, Waldman D. Minimal residual disease in childhood acute lymphoblastic leukemia: current status and challenges. Acta Haematol 2004;112:34–9. [6] Flohr T, Schrauder A, Cazzaniga G, et al. Minimal residual disease-directed risk stratification using real-time quantitative PCR analysis of immunoglobulin and T-cell receptor gene rearrangements in the international multicenter trial AIEOP-BFM ALL 2000 for childhood acute lymphoblastic leukemia. Leukemia 2008;22:771–82. [7] Ryan J, Quinn F, Meunier A, et al. Minimal residual disease detection in childhood acute lymphoblastic leukaemia patients at multiple time-points reveals high levels of concordance between molecular and immunophenotypic approaches. Br J Haematol 2009;144:107–15. [8] Zhou J, Goldwasser MA, Li A, 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 2007;110:1607–11. [9] van Dongen JJ, Seriu T, Panzer-Grumayer ER, et al. Prognostic value of minimal residual disease in acute lymphoblastic leukaemia in childhood. Lancet 1998;352:1731–8. [10] Coustan-Smith E, Behm FG, Sanchez J, et al. Immunological detection of minimal residual disease in children with acute lymphoblastic leukaemia. Lancet 1998;351:550–4. [11] Nyvold C, Madsen HO, Ryder LP, et al. Precise quantification of minimal residual disease at day 29 allows identification of children with acute lymphoblastic leukemia and an excellent outcome. Blood 2002;99:1253–8. [12] Paganin M, Zecca M, Fabbri G, et al. Minimal residual disease is an important predictive factor of outcome in children with relapsed ‘high-risk’ acute lymphoblastic leukemia. Leukemia 2008;22:2193–200. [13] Donovan JW, Ladetto M, Zou G, et al. Immunoglobulin heavy-chain consensus probes for real-time PCR quantification of residual disease in acute lymphoblastic leukemia. Blood 2000;95:2651–8. [14] Coustan-Smith E, Sancho J, Behm FG, et al. Prognostic importance of measuring early clearance of leukemic cells by flow cytometry in childhood acute lymphoblastic leukemia. Blood 2002;100:52–8.

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