Acute Leukemia

Presence of CD34+CD38CD58 leukemia-propagating cells at diagnosis identifies patients at high risk of relapse with Ph chromosome-positive ALL after allo-hematopoietic SCT

Abstract

Relapse of Ph chromosome-positive ALL (Ph+ALL) results from the persistence of leukemia-propagating cells (LPCs). In Ph+ALL, a xenograft assay recently determined that LPCs are enriched in the CD34+CD38CD58 fraction. Therefore, the prognostic significance of LPCs in Ph+ALL subjects after allogeneic hematopoietic SCT (allo-HSCT) was investigated. A total of 80 consecutive adults with Ph+ALL who underwent allo-HSCT were eligible. A multi-parameter flow cytometry analysis examining CD58–FITC/CD10–PE/ CD19–APC–Cy7/CD34–PerCP/CD45–Vioblue/ CD38–APC on gated leukemia BM blasts was performed at diagnosis. Based on the original blast phenotypes, subjects were stratified into the CD34+CD38CD58group (N=15) and other phenotype group (N=65). During minimal residual disease monitoring, significantly higher levels of BCR/ABL transcripts were detected in subjects in the CD34+CD38CD58 group than in other phenotype group, especially at 3 months post HSCT. In addition, CD34+CD38CD58LPCs are directly correlated with a higher 3-year cumulative incidence of relapse (CIR) and worse leukemia-free survival (LFS) and OS. Multivariate analyses indicated that presence of CD34+CD38CD58 LPCs at diagnosis, and BCR–ABL reduction at 3 months post HSCT were independent risk factors for relapse, LFS and OS. Our data suggest that presence of CD34+CD38CD58 LPCs at diagnosis allows rapid identification of high-risk patients for relapse after allo-HSCT.

Introduction

The Ph chromosome, resulting from a reciprocal translocation between chromosomes 9 and 22 and leading to the generation of the BCR–ABL fusion gene, encodes a constitutively active protein tyrosine kinase and is present in 20–30% of adult patients with ALL.1, 2 Despite the widespread use of Abelson tyrosine kinase inhibitors to treat Ph-positive ALL (Ph+ALL), allo-hematopoietic SCT (allo-HSCT) is currently considered the best curative option. Administration of tyrosine kinase inhibitors pre and post transplantation has further improved the prognosis of Ph+ALL. Nevertheless, relapse is still the main cause of treatment failure even after allo-HSCT.1, 3, 4, 5 Once patients experience a relapse, the outcomes are extremely unfavorable. Therefore, it is imperative to identify novel prognostic factors to predict relapse in Ph+ALL after allo-HSCT.

Leukemia-propagating cells (LPCs) are defined by their ability to initiate human leukemia and to proliferate and self-renew in immune-deficient mice.6, 7, 8 Higher LPC frequencies and a gene expression profile typical of LPCs at diagnosis are predictive of unfavorable clinical outcomes in AML.9, 10, 11, 12 Recently, we reported that LPCs in Ph+ALL are enriched in the CD34+CD38CD58 fraction using an anti-CD122-conditioned NOD/SCID xenograft assay by intra-BM injection. Moreover, our preliminary data indicate that a CD34+CD38CD58 LPC phenotype at diagnosis is an independent risk factor for relapse in a cohort of 63 patients with de novo Ph+ALL.13 However, it is unclear if the reliability of the LPC phenotype to identify patients at high risk for relapse is relevant to Ph+ALL patients after allo-HSCT. Consequently, we conducted a cohort study to investigate the impact of the CD34+CD38CD58 LPC phenotype at diagnosis on allo-HSCT outcomes in 80 adults with Ph+ALL.

Materials and methods

Eligibility

Eighty consecutive adults with Ph+ALL who received allo-HSCT at Peking University Institute of Hematology from 1 January 2009 to 31 December 2013 were eligible for this study. The inclusion criteria were as follows: (1) patients were 18–60 years old; (2) diagnosis of Ph+ALL was based on the 2008 WHO (World Health Organization) criteria; (3) allo-HSCT from any source (either HLA-matched sibling donor, HLA-matched unrelated donor or HLA-mismatched/haploidentical donors) was acceptable; and (4) no contraindications to imatinib or an allotransplant. Patients were excluded if either hematological relapse or extramedullary leukemia involvement was diagnosed after initial engraftment or if the life expectancy was <1 month post HSCT. Comprehensive clinical data were available for all subjects (Table 1). The study was approved by the Ethics Committee of Peking University People’s Hospital, and written informed consent was obtained from all subjects before entering the study in accordance with the Declaration of Helsinki.

Table 1 Patient characteristics in the CD34+CD38CD58 and other phenotype groups

Definition of the CD34+CD38CD58LPC phenotype

Multi-parameter flow cytometry analyses of CD58–FITC (Beckman-Coulter, Brea, CA, USA)/CD10–PE/CD19–APC–Cy7/CD34–PerCP/CD45–Vioblue/CD38–APC (BD Biosciences, San Jose, CA, USA) on gated BM leukemia blasts from subjects were performed at diagnosis using a multi-color MACSQuant Analyzer (Miltenyi Biotec, Bergisch Gladbach, Germany). Fluorescence-minus-one controls14, 15 were used to identify positive events for CD34, CD38 and CD58. Samples in which 20% of the blasts expressed the relevant CD Ag were considered positive.16 Based on the blast phenotypes at diagnosis, subjects were further divided into the CD34+CD38CD58group and other phenotype group (including subjects with CD34+CD38CD58+, CD34+CD38+CD58 or CD34+CD38+CD58+ phenotypes and subjects with all of the above defined four fractions) as previously described.13 Analyses were performed using MACSQuant software (Miltenyi Biotec).

Treatment protocols

All of the subjects received uniform first-line imatinib-based induction and consolidation chemotherapy as previously reported.13 After two cycles of consolidation, subjects with a suitable donor, including a matched sibling donor, haploidentical donor or HLA-matched unrelated donor, received an allo-HSCT. The transplant protocols, including donor selection, HLA typing and stem cell harvesting, pre-transplant conditioning regimens and prophylaxis of GVHD, were conducted as described previously in detail.17, 18, 19, 20 Imatinib treatment was scheduled for 3–12 months post HSCT, after the BCR–ABL transcript levels were negative for at least three consecutive tests or major molecular remission (MMR) was sustained for at least 3 months, as described in our previous report.3

Minimal residual disease (MRD) assessment

BCR/ABL transcript levels in BM samples were monitored at diagnosis; directly before transplantation; 1, 2, 3, 6, 9, 12, 24, 36 and 60 months post HSCT; and at relapse using RQ–PCR as previously described.20, 21 In patients with a >1log rising level of BCR/ABL transcripts, monitoring was performed every 2 weeks. BCR/ABL primers and probes amplifying the b3a2 and b2a2 junctions were used for detection according to recommendations of the Europe Against Cancer Program.22, 23 The results were expressed as a transcript ratio and calculated as [fusion gene/ABL] × 100.

Clinical definitions

Hematopoietic engraftment post transplantation was defined as the reconstitution of both neutrophil and plts numbers. Neutrophil reconstitution was defined as occurring after the first 3 consecutive days with an ANC >0.5 × 109/L, and plt reconstitution was defined as the first time levels reached >20 × 109/L for 7 consecutive days. Chimerism analysis was performed using DNA fingerprinting for STRs in blood samples and/or chromosome FISH of BM samples. Full donor chimerism was defined as the absence of detectable recipient hematopoietic or lymphoid cells using either of the above methods. Poor graft function (PGF, both primary and secondary) was defined as the presence of 2 or 3 cytopenic counts (ANC0.5 × 109/L and PLT20 × 109/L or Hb70 g/L) for at least 3 consecutive days beyond 28 days post-transplantation as well as a transfusion requirement associated with hypoplastic-aplastic BM in the presence of complete donor chimerism.24 Secondary PGF was defined as recurrent pancytopenia reaching levels fulfiling the diagnostic criteria for PGF after achieving good graft function in the absence of severe GVHD or hematological relapse. Hematological relapse was determined by the reappearance of blasts in the blood, BM (>5%) or any extramedullary site after CR using common morphological criteria. GVHD was scored as acute or chronic based on published criteria.17, 18, 19, 20

MMR was defined as a 3 log reduction in BCR/ABL transcripts levels compared with the individualized pretreatment baseline. Non-MMR was defined as <3 log reduction in BCR/ABL transcripts levels.

End points and statistical analyses

The primary endpoint was relapse, whereas the secondary endpoints were leukemia-free survival (LFS) and OS. Cumulative incidence of relapse (CIR) and NRM were calculated using the Kalbfleisch and Prentice method. Differences in CIR between the subgroups were assessed according to the Gray test using R software. The probabilities of LFS and OS were estimated using the Kaplan–Meier method and compared using the log-rank test. Univariate analyses were performed using the χ2-test for categorical variables and the Mann–Whitney U-test for continuous variables. Factors at a level of P<0.1 were included as variables in the multivariate Cox regression model. P<0.05 was considered significant unless otherwise specified. The SPSS 16.0 (IBM, Armonk, NY, USA), GraphPad Prism 5.0 (GraphPad Software, La Jolla, CA, USA) and R (Bell Labs, New Providence, NJ, USA) software packages were used for all data analyses. Surviving subjects were accounted for on 1 July 2014.

Results

Overall outcomes

A total of 80 consecutive adults with Ph+ALL who underwent allo-HSCT were enrolled in this study. Imatinib therapy was administered to all of the subjects pre HSCT. Five subjects did not receive imatinib therapy post HSCT for the following reasons: pancytopenia (N=3) or severe gut GVHD (N=2). The median follow-up time was 25.5 months (range, 6–65 months) for all of the subjects and 33 months (range, 6–65 months) for survivors. A total of 77 subjects (96.25%) achieved sustained hematopoietic engraftment. Twelve (15%) subjects relapsed, and 59 (73.75%) remained in continuous CR. Twenty-one subjects died (9 from NRM and 12 from relapse), and 59 subjects survived to the median of 33 months after transplantation (range, 6–65 months). As illustrated in Figure 1, CIR at 3 years was 17.5% (95% confidence interval, 16.03–18.97%), and NRM at 3 years was 13.98% (13.59–14.37%). Three-year probabilities of LFS and OS were 68.51% (55–78.72%) and 72.34% (58.76–82.11%), respectively.

Figure 1
figure1

The CIR, LFS and OS of the entire cohort of Ph+ALL patients. (a) Cumulative incidence of relapse (CIR). (b) Leukemia-free survival (LFS). (c) Overall survival (OS).

Impact of the CD34+CD38CD58 LPC phenotype on clinical outcomes

According to previously reported gating strategies,13 the CD34+CD38CD58 LPC phenotype was detected in 15 subjects at diagnosis, whereas 65 subjects showed the other phenotype. As summarized in Table 1, the demographic and clinical characteristics showed no significant differences between the two phenotypic groups.

Engraftment

Chimerism analysis indicated that all subjects (N=80) achieved full donor chimerism by 30 days post HSCT. Except for the three subjects with secondary PGF in the other phenotype cohort, the cumulative 30-day myeloid engraftment probability and 60-day platelet engraftment probability were 93.33% and 86.67%, respectively, in the CD34+CD38CD58cohort compared with 95.38% and 89.23% in the other phenotype cohort. Moreover, no significant differences were demonstrated in the median time to myeloid engraftment (18 days vs 13 days, P=0.83) and platelet engraftment (21 days vs 15 days, P=0.92) between the CD34+CD38CD58cohort and the other phenotype cohort.

MRD

As shown in Table 1, BCR/ABL transcripts in the CD34+CD38CD58 cohort directly, pre HSCT, did not differ significantly from the transcript levels in the other phenotype cohort (0.09 (0–22.6)% vs 0.04 (0–54.2)%; P=0.56). Significantly higher levels of BCR/ABL transcripts were detected in subjects from the CD34+CD38CD58 cohort compared with subjects in the other phenotype cohort at 2 months post HSCT (0.01 (0–50.8)% vs 0 (0–96.8)%; P=0.02) and, in particular, at 3 months post HSCT (0.12 (0–152.4)% vs 0 (0–100)%; P=0.001).

Relapse

In the CD34+CD38CD58 cohort, 8 of 15 subjects who underwent HSCT relapsed compared with 4 of 65 subjects in the other phenotype cohort (P<0.0001). Relapse occurred at a median of 13 months (3–28 months) post transplantation in the CD34+CD38CD58 cohort and at 14.5 months (6–45 months) in the other phenotype cohort. CIR at 3 years in the CD34+CD38CD58 cohort was significantly higher compared with CIR in the other phenotype cohort (63.2% (58.2–68.1%) vs 5.3% (5.1–5.5%); P<0.0001; Figure 2a). Other variables significantly associated with CIR in univariate analyses included MMR at 3 months post HSCT (P<0.0001) and disease status pre HSCT (P<0.0001). The LPC phenotype at diagnosis and MMR at 3 months post HSCT were independently correlated with relapse in the multivariate analyses (Table 2).

Figure 2
figure2

The CIR, LFS and OS of the groups of Ph+ALL patients undergoing allo-HSCT (N=80) according to the CD34+CD38CD58 LPC or other phenotype at diagnosis. (a) Cumulative incidence of relapse (CIR). (b) Leukemia-free survival (LFS). (c) Overall survival (OS).

Table 2 Univariate and multivariate analyses of relapse, LFS and OS in Ph+ALL patients undergoing allo-HSCT (N=80)

Leukemia-free survival

The three-year LFS of subjects in the other phenotype cohort was significantly higher than in subjects in the CD34+CD38CD58 cohort (78.7% (64.5–87.7%) vs 30.2% (8.1–56.6%); P=0.001; Figure 2b). MMR at 3 months post HSCT (P<0.0001) and disease status pre HSCT (P<0.0001) were also significantly correlated with LFS in the univariate analyses. In multivariate analyses, the LPC phenotype at diagnosis and MMR at 3 months post HSCT were independently correlated with LFS (Table 2).

OS

The CD34+CD38CD58 cohort exhibited poorer 3-year OS than the other phenotype cohort (37.7% (12.6–63.2%) vs 82.3% (68.5–90.4%); P=0.0004; Figure 2c). MMR at 3 months post HSCT (P<0.0001) and disease status pre HSCT (P<0.0001) were correlated with survival in the univariate analyses. In multivariate analyses, the LPC phenotype at diagnosis and MMR at 3 months post HSCT were independently correlated with OS (Table 2).

A new risk stratification model was established by integrating the analysis of the CD34+CD38CD58 LPC phenotype at diagnosis and MMR at 3 months post HSCT

Based on the multivariate analyses, three distinct prognostic groups (the LPC+ and non-MMR group, the other group (LPC and non-MMR or LPC+ and MMR) and the LPC and MMR group) were further classified by integration of the two independent risk factors, including the CD34+CD38CD58 phenotype at diagnosis (LPC+=CD34+CD38CD58 phenotype, LPC=other phenotype) and MMR at 3 months post HSCT (non-MMR= BCR–ABL reduction<3 log, MMR=BCR–ABL reduction3 log). These groups significantly showed different 3-year CIR values (100% vs 49.35% (41.53–51.17%) vs 5.47% (5.27–5.67%); P<0.0001; Figure 3a), LFS (0% vs 37.98% (9.71–66.91%) vs 80.20% (66–88.95%); P<0.0001; Figure 3b) and OS (0% vs 50.35% (17.04–76.64%) vs 83.86% (70.02–91.67%); P<0.0001; Figure 3c), respectively.

Figure 3
figure3

A new risk stratification model was established by the combined analysis of the CD34+CD38CD58 LPC phenotype at diagnosis (LPC+=CD34+CD38CD58 phenotype, LPC=other phenotype) and BCR-ABL reduction at 3 months post HSCT (non-MMR= BCR–ABL reduction<3 log, MMR=BCR–ABL reduction3 log). (a) Cumulative incidence of relapse (CIR). (b) Leukemia-free survival (LFS). (c) Overall survival (OS).

Discussion

To our knowledge, our current cohort study is the first to demonstrate that the presence of CD34+CD38CD58 LPCs at diagnosis is significantly correlated with a higher MRD frequency and a 3-year CIR in Ph+ALL patients after allo-HSCT. Prospective evaluation of CD34+CD38CD58 LPCs at diagnosis might allow the identification of subgroups of Ph+ALL transplant patients at high risk for relapse.

Relapse remains one of the major obstacles to Ph+ALL treatment after allo-HSCT, with an estimated 4-year relapse of 38% in a UKALL12/E2993 study.4 In a Japanese adult leukemia study, an estimated 3-year CIR of 21% was observed in an imatinib cohort, which was significantly lower than that in the non-imatinib cohort (34%).5 Our previous study demonstrated that patients treated with imatinib maintenance therapy guided by BCR-ABL monitoring by RQ–PCR after allo-HSCT have a lower estimated 5-year relapse rate (10.2%) compared with patients not treated with imatinib (33.1%).3 However, challenges remain in implementing the experimentally validated biomarkers for recurrence prediction for risk-stratification therapy in Ph+ALL after allo-HSCT. In agreement with our previous report for de novoPh+ALL,13 the novel CD34+CD38CD58LPC phenotype has significant power to identify patients at high risk for relapse post HSCT. Although allo-HSCT exhibits the strongest anti-leukemia effects, it could not completely overcome the high risk of relapse in adult Ph+ALL patients with the candidate LPC phenotype. Assessment of the CD34+CD38CD58 LPC phenotype can be easily incorporated into routine diagnostic protocols to predict recurrence and guide individualized post-transplantation therapy. We are aware, however, that this is a single cohort study enrolling 80 patients with Ph+ALL after allo-HSCT. This approach requires prospective validation in a multicenter prospective cohort study with more patients.

For Ph+ALL outcomes, other prognostic factors have been proposed, such as BCR/ABL-based MRD monitoring. Reports from the pre-tyrosine kinase inhibitor era suggested a strong correlation between BCR/ABL quantification and recurrence.25, 26 However, it remains controversial whether BCR/ABL-based MRD monitoring pre and post HSCT continues to serve as an efficient tool for risk stratification after HSCT.27, 28, 29, 30 Our study demonstrates the prognostic relevance of MRD kinetics to relapse and strongly supports a prior report from the GMALL Study Group,28 which found that patients with the appearance of BCR/ABL transcripts early (<100 days) and/or at higher levels (>10−3) after allo-HSCT show a limited benefit from imatinib treatment. Unlike a previous study by Lee et al.,27 our data indicate that MRD status pre HSCT is not significantly correlated with recurrence post HSCT. An insufficient molecular response pre HSCT suggests that although first-line imatinib-based chemotherapy is highly active against leukemic bulk, it is not effective against the LPCs responsible for relapse.31, 32 It is therefore conceivable that subsequent allo-HSCT might partially compensate for this insufficiency due to the GVL effect; therefore, MRD kinetics post HSCT might be more closely associated with the outcomes. Remarkably, a new risk stratification model is further established by the combined analyses of pre-HSCT (CD34+CD38CD58 LPCs at diagnosis) and post-HSCT parameters (MMR at 3 months post HSCT), which is helpful to identify a small subset of patients at ‘very high risk’ of relapse (100%) post transplantation. Consequently, rapid identification of high-risk patients based on the pre-HSCT parameter(CD34+CD38CD58 LPCs at diagnosis) might allow patients to be treated with prophylactic interventions such as new tyrosine kinase inhibitors,33, 34 DLI19, 35 or interferon alpha36, 37 early after allo-HSCT for the prevention of relapse and the improvement of transplant outcomes in adult Ph+ALL.

IKZF1-deletions were recently identified to correlate with relapse in Ph+ALL.38, 39 In the present cohort study, IKZF1-deletions were analyzed in 52.5% of the subjects, which made it difficult to directly compare the impact of the LPC phenotype or IKZF1-deletions on post-HSCT outcomes. It should be noted, however, that our preliminary results demonstrated that the frequency of IKZF1-deletions at diagnosis were comparable between the two phenotypic groups, which emphasizes the prognostic significance of the LPC phenotype. In the future, it would be of value to investigate the two risk factors in prospective randomized studies with larger patient cohorts.

In conclusion, our data suggest that the presence of CD34+CD38CD58 LPCs at diagnosis allows rapid identification of high-risk patients of relapse after allo-HSCT. Risk-stratification post-HSCT therapy, incorporating an analysis of the CD34+CD38CD58 LPC phenotype at diagnosis, shows promise to benefit adults with Ph+ALL in the future.

References

  1. 1

    Fielding AK . How I treat Philadelphia chromosome-positive acute lymphoblastic leukemia. Blood 2010; 116: 3409–3417.

  2. 2

    Pui CH, Robison LL, Look AT . Acute lymphoblastic leukaemia. Lancet 2008; 371: 1030–1043.

  3. 3

    Chen H, Liu KY, Xu LP, Liu DH, Chen YH, Zhao XY et al. Administration of imatinib after allogeneic hematopoietic stem cell transplantation may improve disease-free survival for patients with Philadelphia chromosome-positive acute lymphobla stic leukemia. J Hematol Oncol 2012; 5: 29.

  4. 4

    Fielding AK, Rowe JM, Buck G, Foroni L, Gerrard G, Litzow MR et al. UKALLXII/ECOG2993: addition of imatinib to a standard treatment regimen enhances long-term outcomes in Philadelphia positive acute lymphoblastic leukemia. Blood 2014; 123: 843–850.

  5. 5

    Mizuta S, Matsuo K, Nishiwaki S, Imai K, Kanamori H, Ohashi K et al. Pretransplant administration of imatinib for allo-HSCT in patients with BCR-ABL-positive acute lymphoblastic leukemia. Blood 2014; 123: 2325–2332.

  6. 6

    Valent P, Bonnet D, De Maria R, Lapidot T, Copland M, Melo JV et al. Cancer stem cell definitions and terminology: the devil is in the details. Nat Rev Cancer 2012; 12: 767–775.

  7. 7

    Lapidot T, Sirard C, Vormoor J, Murdoch B, Hoang T, Caceres-Cortes J et al. A cell initiating human acute myeloid leukaemia after transplantation into SCID mice. Nature 1994; 367: 645–648.

  8. 8

    Kong Y, Yoshida S, Saito Y, Doi T, Nagatoshi Y, Fukata M et al. CD34+CD38+CD19+ as well as CD34+CD38-CD19+ cells are leukemia-initiating cells with self-renewal capacity in human B-precursor ALL. Leukemia 2008; 22: 1207–1213.

  9. 9

    Eppert K, Takenaka K, Lechman ER, Waldron L, Nilsson B, van Galen P et al. Stem cell gene expression programs influence clinical outcome in human leukemia. Nat Med 2011; 17: 1086–1093.

  10. 10

    Gerber JM, Smith BD, Ngwang B, Zhang H, Vala MS, Morsberger L et al. A clinically relevant population of leukemic CD34(+)CD38(−) cells in acute myeloid leukemia. Blood 2012; 119: 3571–3577.

  11. 11

    van Rhenen A, Feller N, Kelder A, Westra AH, Rombouts E, Zweegman S et al. High stem cell frequency in acute myeloid leukemia at diagnosis predicts high minimal residual disease and poor survival. Clin Cancer Res 2005; 11: 6520–6527.

  12. 12

    Gentles AJ, Plevritis SK, Majeti R, Alizadeh AA . Association of a leukemic stem cell gene expression signature with clinical outcomes in acute myeloid leukemia. JAMA 2010; 304: 2706–2715.

  13. 13

    Kong Y, Chang YJ, Liu YR, Wang YZ, Jiang Q, Jiang H et al. CD34+CD38CD58 cells are leukemia-propagating cells in Philadelphia chromosome-positive acute lymphoblastic leukemia. Leukemia 2014 (epub ahead of print 25 July 2014; doi:10.1038/leu.2014.228).

  14. 14

    Baumgarth N, Roederer M . A practical approach to multicolor flow cytometry for immunophenotyping. J Immunol Methods 2000; 243: 77–97.

  15. 15

    Bayer J, Grunwald D, Lambert C, Mayol JF, Maynadie M . Thematic workshop on fluorescence compensation settings in multicolor flow cytometry. Clin Cytom 2007; 72: 8–13.

  16. 16

    Bene MC, Castoldi G, Knapp W, Ludwig WD, Matutes E, Orfao A et al. Proposals for the immunological classification of acute leukemias. Leukemia 1995; 9: 1783–1786.

  17. 17

    Xiao-Jun H, Lan-Ping X, Kai-Yan L, Dai-Hong L, Yu W, Huan C et al. Partially matched related donor transplantation can achieve outcomes comparable with unrelated donor transplantation for patients with hematologic malignancies. Clin Cancer Res 2009; 15: 4777–4783.

  18. 18

    Huang XJ, Zhu HH, Chang YJ, Xu LP, Liu DH, Zhang XH et al. The superiority of haploidentical related stem cell transplantation over chemotherapy alone as postremission treatment for patients with intermediate- or high-risk acute myeloid leukemia in first complete remission. Blood 2012; 119: 5584–5590.

  19. 19

    Yan CH, Liu DH, Liu KY, Xu LP, Liu YR, Chen H et al. Risk stratification-directed donor lymphocyte infusion could reduce relapse of standard-risk acute leukemia patients after allogeneic hematopoietic stem cell transplantation. Blood 2012; 119: 3256–3262.

  20. 20

    Wang Y, Wu DP, Liu QF, Qin YZ, Wang JB, Xu LP et al. RUNX1/RUNX1T1-based MRD-monitoring early after allogeneic transplantation rather than c-KIT mutations in adult t(8;21) AML allows further risk stratification. Blood, (e-pub ahead of print 31 July 2014; pii: blood-2014-03-563403).

  21. 21

    Qin YZ, Liu YR, Zhu HH, Li JL, Ruan GR, Zhang Y et al. Different kinetic patterns of BCR-ABL transcript levels in imatinib-treated chronic myeloid leukemia patients after achieving complete cytogenetic response. Int J Lab Hematol 2008; 30: 317–323.

  22. 22

    Beillard E, Pallisgaard N, van der Velden VH, Bi W, Dee R, van der Schoot E et al. Evaluation of candidate control genes for diagnosis and residual disease detection in leukemic patients using 'real-time' quantitative reverse-transcriptase polymerase chain reaction (RQ-PCR) - a Europe against cancer program. Leukemia 2003; 17: 2474–2486.

  23. 23

    Gabert J, Beillard E, van der Velden VH, Bi W, Grimwade D, Pallisgaard N et al. Standardization and quality control studies of 'real-time' quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia-a Europe Against Cancer program. Leukemia 2003; 17: 2318–2357.

  24. 24

    Kong Y, Chang YJ, Wang YZ, Chen YH, Han W, Wang Y et al. Association of an impaired bone marrow microenvironment with secondary poor graft function after allogeneic hematopoietic stem cell transplantation. Biol Blood Marrow Transplant 2013; 19: 1465–1473.

  25. 25

    Preudhomme C, Henic N, Cazin B, Lai JL, Bertheas MF, Vanrumbeke M et al. Good correlation between RT-PCR analysis and relapse in Philadelphia (Ph1)-positive acute lymphoblastic leukemia (ALL). Leukemia 1997; 11: 294–298.

  26. 26

    Pane F, Cimino G, Izzo B, Camera A, Vitale A, Quintarelli C et al. Significant reduction of the hybrid BCR/ABL transcripts after induction and consolidation therapy is a powerful predictor of treatment response in adult Philadelphia-positive acute lymphoblastic leukemia. Leukemia 2005; 19: 628–635.

  27. 27

    Lee S, Kim YJ, Chung NG, Lim J, Lee DG, Kim HJ et al. The extent of minimal residual disease reduction after the first 4-week imatinib therapy determines outcome of allogeneic stem cell transplantation in adults with Philadelphia chromosome-positive acute lymphoblastic leukemia. Cancer 2009; 115: 561–570.

  28. 28

    Pfeifer H, Wassmann B, Bethge W, Dengler J, Bornhauser M, Stadler M et al. Randomized comparison of prophylactic and minimal residual disease-triggered imatinib after allogeneic stem cell transplantation for BCR-ABL1-positive acute lymphoblastic leukemia. Leukemia 2013; 27: 1254–1262.

  29. 29

    Wassmann B, Pfeifer H, Stadler M, Bornhauser M, Bug G, Scheuring UJ et al. Early molecular response to posttransplantation imatinib determines outcome in MRD+ Philadelphia-positive acute lymphoblastic leukemia (Ph+ALL). Blood 2005; 106: 458–463.

  30. 30

    Yanada M, Sugiura I, Takeuchi J, Akiyama H, Maruta A, Ueda Y et al. Prospective monitoring of BCR-ABL1 transcript levels in patients with Philadelphia chromosome-positive acute lymphoblastic leukaemia undergoing imatinib-combined chemotherapy. Bri J Haematol 2008; 143: 503–510.

  31. 31

    Copland M, Hamilton A, Elrick LJ, Baird JW, Allan EK, Jordanides N et al. Dasatinib (BMS-354825) targets an earlier progenitor population than imatinib in primary CML but does not eliminate the quiescent fraction. Blood 2006; 107: 4532–4539.

  32. 32

    Saito Y, Kitamura H, Hijikata A, Tomizawa-Murasawa M, Tanaka S, Takagi S et al. Identification of therapeutic targets for quiescent, chemotherapy-resistant human leukemia stem cells. Sci Transl Med 2010; 2: 17ra9.

  33. 33

    Talpaz M, Shah NP, Kantarjian H, Donato N, Nicoll J, Paquette R et al. Dasatinib in imatinib-resistant Philadelphia chromosome-positive leukemias. N Engl J Med 2006; 354: 2531–2541.

  34. 34

    Cortes JE, Kantarjian H, Shah NP, Bixby D, Mauro MJ, Flinn I et al. Ponatinib in refractory Philadelphia chromosome-positive leukemias. N Engl J Med 2012; 367: 2075–2088.

  35. 35

    Savani BN, Srinivasan R, Espinoza-Delgado I, Dorrance C, Takahashi Y, Igarashi T et al. Treatment of relapsed blast-phase Philadelphia-chromosome-positive leukaemia after non-myeloablative stem-cell transplantation with donor lymphocytes and imatinib. Lancet Oncol 2005; 6: 809–812.

  36. 36

    Visani G, Martinelli G, Piccaluga P, Tosi P, Amabile M, Pastano R et al. Alpha-interferon improves survival and remission duration in P-190BCR-ABL positive adult acute lymphoblastic leukemia. Leukemia 2000; 14: 22–27.

  37. 37

    Wassmann B, Scheuring U, Pfeifer H, Binckebanck A, Kabisch A, Lubbert M et al. Efficacy and safety of imatinib mesylate (Glivec) in combination with interferon-alpha (IFN-alpha) in Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL). Leukemia 2003; 17: 1919–1924.

  38. 38

    Martinelli G, Iacobucci I, Storlazzi CT, Vignetti M, Paoloni F, Cilloni D et al. IKZF1 (Ikaros) deletions in BCR-ABL1-positive acute lymphoblastic leukemia are associated with short disease-free survival and high rate of cumulative incidence of relapse: a GIMEMA AL WP report. J Clin Oncol 2009; 27: 5202–5207.

  39. 39

    Mullighan CG, Miller CB, Radtke I, Phillips LA, Dalton J, Ma J et al. BCR-ABL1 lymphoblastic leukaemia is characterized by the deletion of Ikaros. Nature 2008; 453: 110–114.

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (grant no. 81370638 & 81230013), the Beijing Municipal Science and Technology Program, the National Clinical Priority Specialty and the Peking University People’s Hospital Research and Development Funds (grant no. RDB2012-23). American Journal Experts (www.journalexperts.com) offered editorial assistance to the authors during the preparation of this manuscript. We thank all of the core facilities at the Peking University Institute of Hematology for sample collection.

Author Contributions

X-JH designed the study and supervised the analyses and manuscript preparation. YK performed the research, analyzed and interpreted the data, performed statistical analyses and wrote the manuscript. All other authors participated in the collection of patient data. All the authors agreed to submit the final manuscript.

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Correspondence to X-J Huang.

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