Immunophenotype

Detection of acute leukemia cells with mixed lineage leukemia (MLL) gene rearrangements by flow cytometry using monoclonal antibody 7.1

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Abstract

Translocations involving 11q23 are among the most common genetic abnormalities in hematologic malignancies, occurring in approximately 5–10% of acute lymphoblastic leukemia (ALL) and 5% of acute myeloblastic leukemia (AML). In 11q23 translocations, the mixed lineage leukemia (MLL) gene on chromosome 11, band q23, is usually disrupted. The human homologue of the rat NG2 chondroitin sulfate proteoglycan molecule, as detected by the monoclonal antibody (moab) 7.1, was shown to be expressed on leukemic cells with MLL rearrangements of children with acute leukemia. We further investigated the reactivity of the moab 7.1 on 533 cell samples of adults (n = 215) and children (n = 318) with acute leukemias (271 AML, 217 B-lineage ALL, 37 T-lineage ALL, eight CD7+ CD56+ myeloid/natural killer cell precursor acute leukemias) by flow cytometry. In AML, 38 samples were positive for moab 7.1 (‘20%-cut-off-level’). These moab 7.1-positive AML cases revealed a myelomonocytic-differentiated immunophenotype with coexpression of the NK cell marker CD56 in 33 of 38 cases. Two of eight cell samples of the recently described CD7+ CD56+ myeloid/natural killer cell precursor acute leukemia entity reacted with moab 7.1. In ALL, 35 samples mostly of the pro-B-ALL subtype (33 pro-B-ALL, one common-ALL, one pre-B-ALL) were positive for moab 7.1. 58 (81%) of 72 samples with MLL rearrangements were positive for moab 7.1 including 28/31 with a t(4;11), 16/17 with a t(9;11), 3/5 with a t(11;19), and 2/6 with a del(11)(q23). All moab 7.1-positive ALL (n = 34) and childhood AML (n = 17) cases revealed MLL rearrangements as detected by Southern blot analysis and RT-PCR. However, 11 adults with AML, and one adult with moab 7.1-positive CD7+ CD56+myeloid/natural killer cell precursor acute leukemia were negative for MLL rearrangements as proved by Southern blot analysis. We conclude that moab 7.1 is a sensitive but not entirely specific marker for the identification of 11q23-associated AML and ALL by flow cytometry in children and adults.

Introduction

The monoclonal antibody (moab) 7.1 was generated during efforts to identify antigens that may be shared by immature hematopoietic precursor and stroma cells. It recognizes a 220- to 240-kDa cell surface chondroitin sulfate homologue of the rat NG2-surface molecule.1 Moab 7.1 does not react with normal hematopoietic precursor cells or mature blood cells but was shown to specifically react with acute leukemia cells of childhood AML and ALL patients with 11q23 anomalies.123 Translocations involving 11q23 are among the most common genetic abnormalities in hematologic malignancies occurring in approximately 5–10% of acute lymphoblastic leukemia (ALL) as well as 5% of acute myeloblastic leukemia (AML), and have been associated with a poor response to therapy and a dismal prognosis in childhood ALL.456789

In 11q23 translocations, the mixed lineage leukemia (MLL) gene on chromosome 11, band q23, is usually disrupted. MLL is believed to be involved in the regulation of differentiation pathways, however, its exact function is not yet clear.5 The MLL gene is thought to play an important role in leukemogenesis as it has been implicated in fusions with more than 25 other genes in both ALL and AML.10

The moab 7.1-specific antigen NG2 is a member of the chondroitin sulfate proteoglycan family.11 The physiological function of NG2 is largely unknown. NG2 is thought to be involved in cell adhesion and migration mechanisms, mainly in the central nervous system. It is known that NG2 is highly expressed in early embryonic development and downregulated during differentiation.1213 It was assumed that NG2 is usually expressed at an early stage of embryonic hematopoietic development and that MLL rearrangements may lead to an aberrant continued expression of NG2.1

Up to now, only leukemic cells of children with ALL or AML have been examined for their reactivity with moab 7.1. In childhood ALL, a 100% sensitivity and specificity for leukemic cells with MLL rearrangements has been observed.2 In contrast, the sensitivity of moab 7.1 in childhood AML for leukemic cells with MLL rearrangements was 54–78% despite a 100% specificity.13 To our knowledge, no data concerning the reactivity of moab 7.1 with leukemic cells of adults with acute leukemia have been reported so far. We therefore investigated a large series of cell samples of adults and children with acute leukemia to further determine the value of moab 7.1 in the detection of acute leukemia cells with MLL rearrangements by flow cytometry.

Material and methods

Patients and cell samples

We examined either freshly obtained or cryopreserved bone marrow or peripheral blood samples of 533 patients with acute leukemia for their reactivity with moab 7.1. In our series, 238 children with ALL (204 B-lineage ALL, 34 T-lineage ALL), 78 children with AML, 193 adults with AML, and 16 adults with ALL (13 B-lineage ALL, three T-lineage ALL) were included. Moreover, eight cell samples of patients (six adults, two children) with the recently described CD7+ CD56+ myeloid/natural killer cell precursor acute leukemia phenotype14 were analyzed for moab 7.1 reactivity. Diagnosis of acute leukemia was based on immunophenotypical criteria according to the EGIL recommendations15 and morphologically in accordance with the FAB criteria.1617 B-lineage ALL cases were subclassified into pro-B-ALL (n = 53), common-ALL (n = 113), pre-B-ALL (n = 43), and mature B-ALL (n = 5) according to the EGIL guidelines.15 Diagnosis of CD7+ CD56+ myeloid/natural killer cell precursor leukemia was made according to the proposals of Suzuki et al.14 No difference in the expression patterns between freshly obtained and cryopreserved cell samples was observed.

Immunophenotyping

Leukemic cells from heparinized fresh bone marrow or peripheral blood samples were isolated by Ficoll–Hypaque (Pharmacia, Freiburg, Germany) density gradient centrifugation, and leukemia-associated antigens were detected by a panel of moabs either by a direct or an indirect immunofluorescence assay as previously described.1819 Cell samples were analyzed by flow cytometry (FACScan; Becton Dickinson, San Diego, CA, USA) using the Cell-Quest software program (Becton Dickinson). Cell samples were considered positive for a specific antigen if the antigen was expressed on at least 20% of the leukemic cells (‘20-cut-off-level’).181920

Detection of moab 7.1 reactivity in acute leukemia by flow cytometry

Cells were stained by indirect immunofluorescence using moab 7.1 (IgG1; Immunotech, Marseille, France) and FITC-labelled goat-anti-mouse antibody (Dianova, Hamburg, Germany) as a second layer. Non-specific binding of Fcγ-receptors was blocked by preincubation of the cells with a polyclonal rabbit serum (Gibco BRL, Paisley, UK). At least 10 000 cells per sample were acquired and analyzed by flow cytometry as described above. Non-viable cells were excluded from analysis by propidium iodide staining (Sigma, Deisenhofen, Germany). To compare our results with previous studies on moab 7.1 reactivity, cell samples were considered moab 7.1 positive if at least 20% of the cells stained with moab 7.1 revealed a fluorescence intensity higher than 98% of the cells stained with isotype-matched control antibody (‘20-cut-off-level’).123 To avoid false negative results, we evaluated all cases by accurate light scatter gating as well as by subtraction of mature CD3+ T cells and mature surface Ig+ B cells.

Southern blot analysis

Each 5–10 μg DNA was digested with the restriction enzymes BamHI and EcoRI overnight. Completely digested DNA was size fractionated on 0.7% agarose gels and transferred to nylon membranes (SureBlot; Oncor, Gaithersburg, MD, USA). A diogixin-dUTP-labelled MLL cDNA probe (Oncor) containing exons 8–15 was used for hybridization. The membranes were washed under high stringency (0.1 × SSC for 2 × 15 min at 65°C). Detection was performed with an anti-digoxin antibody (Boehringer, Mannheim, Germany) and CSPD (Boehringer).

Nucleic acid isolation and RT-PCR

DNA was extracted with a salting out procedure21 from fresh bone marrow or peripheral blood cells after Ficoll separation of mononucleated cells. From the same specimens total RNA was isolated with the RNeasy kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. One μg of total RNA was reverse transcribed with 200 U Superscript (Gibco BRL) in a 40 μl reaction using random primers. An equivalent quantity of 25 ng RNA amplified for 35 cycles (1 min 94°C, 1 min 60°C, 1 min 72°C), in 50 μl with 10 pmol of each forward primer and reverse primer (primers were described in Ref. 22) 10 mmol dNTPs, and 1.25 units of Taq polymerase (Gibco BRL) in the buffer shipped by the supplier. For each RNA sample an ABL-specific RT-PCR was performed to control the integrity of RNA using primers abl5′: 5′GGCCAG TAGCATCTGACTTTG3′ and abl3′: 5′ATGGTACCAG GAGTGTTTCTCC3′. Water instead of cDNA was included as a blank sample in each experiment. Amplification products were analyzed on 1.5% agarose gels stained with ethidium bromide.

Cytogenetics

Chromosome analyses were performed on metaphases from short-term (24 h, 48 h) cultures of pretreatment bone marrow and/or peripheral blood cells. Cell cultivation and chromosome preparation was carried out according to standard protocols. G-banding was used and the chromosomes interpreted according to ISCN nomenclature.23

Statistical analysis

Differences in the immunophenotypical of expression patterns between moab 7.1-positive and -negative cell samples were analyzed using chi-square test (Pearson coefficient). P values <0.05 were considered significant. The sensitivity of moab 7.1 in the detection of leukemia cells with MLL rearrangements was calculated as follows: {moab 7.1 pos. MLL rearrangements pos. cases/(moab 7.1 pos. MLL rearrangement pos. cases + moab 7.1 neg. MLL rearrangements pos. cases)} × 100%. The positive predictive value of moab 7.1 in the detection of leukemia cells with MLL rearrangements was calculated as follows: {moab 7.1 pos. MLL rearrangement pos. cases/(moab 7.1 pos. MLL rearrangement pos. cases + moab 7.1 pos. MLL rearrangement neg. cases)} × 100%. All statistical analyses were done with the SPSS software program.

Results

Reactivity of moab 7.1 with acute leukemia cells

Seventy-five of the 533 cell samples examined were positive for moab 7.1 (‘20%-cut-off-level’). In these cases, moab 7.1 reacted with 21 to 87% of the leukemic cells (median 46%). Three hundred and eighteen cases were negative for moab 7.1, in 124 cases moab 7.1 reacted with 1 to 9% of the cells, in 16 cases 10 to 19% of the leukemic cells reacted with moab 7.1. Figure 1 shows three representative examples of moab 7.1-positive acute leukemias.

Figure 1
figure1

 Three representative examples of moab 7.1-positive acute leukemias: (a) pro-B-ALL with MLL rearrangement, (b) AML FAB M5 with MLL rearrangement and (c) CD7+ CD56+ myeloid/natural killer cell precursor acute leukemia without MLL rearrangement. The dotted lines represent the specific negative controls, the bold lines the leukemic cells stained with moab 7.1.

Moab 7.1 reactivity in acute leukemia: correlation with immunophenotype and FAB morphology

In ALL, 33 of 53 pro-B-ALL, one of 113 common-ALL, and one of 43 pre-B-ALL cell samples analyzed were positive for moab 7.1 (‘20%-cut-off-level’). All T-lineage ALL cases (n = 37) examined were negative for moab 7.1. Within the pro-B-ALL subtype, moab 7.1-positive cases expressed less CD54 (P < 0.00001), less CD33 (P < 0.0008), less CD13 (P < 0.5), and less cytoplasmic CD22 (P < 0.04) than moab 7.1-negative pro-B-ALL cases (Table 1). In AML, moab 7.1-positive cases revealed a more mature, myelomonocytic-differentiated immunophenotype as characterized by the finding that moab 7.1-positive AML cases expressed more often CD36 (P < 0.00001), CD4 (P < 0.00001), CD56 (P < 0.00001), CD64 (P < 0.0002), CD65s (P < 0.00007), HLA-DR (P < 0.002) and less often myeloperoxidase (P < 0.00001), CD13 (P < 0.00001), CD34 (P < 0.00001), CD117 (P < 0.04) than moab 7.1-negative AML cases (Table 2). Moab 7.1-positive AML cases mostly revealed a myelomonocytic-differentiated morphologic FAB subtype (M5 n = 21, M4 n = 5, M2 n = 2, M1 n = 1, and M0 n = 2). Two of eight CD7+ CD56+ myeloid/natural killer cell precursor acute leukemias were positive for moab 7.1. No clear-cut differences in the immunophenotype of moab 7.1-positive acute leukemia cell samples between children and adults, neither in ALL nor AML, were observed (data not shown).

Table 1  Immunophenotype of moab 7.1-positive and moab 7.1-negative pro-B-ALL cases: positive cell samples/cell samples examined (positive cell samples in %)
Table 2  Immunophenotype of moab 7.1-positive and moab 7.1-negative AML cases: positive cell samples/cell samples examined (positive cell samples in %)

MLL rearrangements and moab 7.1 reactivity

Sensitivity:

Fifty-eight (81%) of 72 samples with MLL rearrangements as demonstrated by Southern blot analysis and RT-PCR were positive for moab 7.1. Correlation with cytogenetic data of the MLL rearrangement-positive leukemic cell samples revealed that 28 of 31 samples with a t(4;11), 16 of 17 samples with a t(9;11), three of five samples with a t(11;19), and two of six samples with a del(11)(q23) were positive for moab 7.1. In childhood ALL, moab 7.1 detected 32 (86%) of 37 cell samples with MLL rearrangements. Two adult patients with pro-B-ALL and MLL rearrangements were examined, and both were found to be positive for moab 7.1. In childhood AML, 17 (77%) of 22 cell samples with MLL rearrangements reacted with moab 7.1. Seven (64%) of 11 adults with AML and MLL rearrangements were positive for moab 7.1. Thus, in our series, the overall sensitivity of moab 7.1 in the detection of leukemic cells with MLL rearrangements was 87% (34/39) in ALL and 73% (24/33) in AML (Table 3).

Table 3  Sensitivity and positive predictive value of moab 7.1 for the detection of MLL rearrangements in acute leukemia by flow cytometry

Positive predictive value:

Thirty-four of 35 moab 7.1-positive ALL (32 children, three adults) had proven MLL rearrangements. Unfortunately, no cell material for Southern blot analysis was available for one moab 7.1-positive common ALL. In AML, all 17 moab 7.1-positive children with AML had MLL rearrangements, whereas only seven of 18 adults with moab 7.1-positive AML revealed MLL rearrangements by Southern blot analysis. No cell material for Southern blot analysis was available for three moab 7.1-positive AML samples. One moab 7.1-positive CD7+ CD56 myeloid/natural killer cell precursor acute leukemia sample was negative for a MLL rearrangement. No cell material for Southern blot analysis was available for the second moab 7.1-positive CD7+ CD56+ myeloid/natural killer cell precursor acute leukemia sample. Therefore, in our series, the positive predictive value of moab 7.1 in the detection of acute leukaemia cells with MLL rearrangement was 100% (32/32) in childhood ALL, 100% (2/2) in adult ALL, 100% (17/17) in childhood AML, and 39% (7/18) in adult AML (Table 3)

Discussion

In this study, we investigated the sensitivity and positive predictive value of the moab 7.1 in the detection of acute leukemia cells with MLL rearrangements in 533 children and adults with acute leukemia.

Previous studies revealed a positive predictive value of 100% and a 54–78% sensitivity in childhood AML.13 In accordance with these data, we observed a positive predictive value of 100% (17/17) and a 77% (17/22) sensitivity in childhood AML. In newly diagnosed childhood ALL, a positive predictive value of 100% and a 100% sensitivity was previously observed.2 Despite a positive predictive value of 100% (32/32), only a 86% (32/37) sensitivity in our series of childhood ALL was seen. Interestingly, Behm et al2 had previously reported a child with relapsed t(4;11)-positive ALL and MLL rearrangement, whose leukemic cells failed to react with moab 7.1. Another reason for the lower sensitivity observed in our series could be the higher number of childhood ALL cell samples with MLL rearrangements analyzed (n = 37) compared to the previous study (n = 9).2 We conclude that moab 7.1 is a highly specific but not absolutely sensitive marker for leukemic cells with MLL rearrangement in childhood acute leukemia.

To our knowledge, no data concerning the reactivity of moab 7.1 with acute leukemia cells of adults have been reported so far. Leukemic cell samples with MLL rearrangements of two adults with t(4;11)-positive pro-B-ALL and seven of 11 adults with AML were positive for moab 7.1. However, 11 cell samples of adults with AML and one cell sample of an adult with a myeloid/natural killer cell precursor immunophenotype reacted with moab 7.1 despite the absence of MLL rearrangements as proved by Southern blot analysis. These results suggest that in adult AML, other transforming events apart from MLL rearrangements may also lead to an aberrant expression of the moab 7.1-specific antigen NG2 in leukemic cells. It was speculated that NG2 is usually expressed at an early stage of embryonic hematopoietic development and MLL rearrangements may lead to an aberrant continued expression of NG2.1 Our finding that NG2 is expressed on leukemic cells of children and adults with acute leukemia raises the question whether the disruption of the MLL gene rather lead to an altered de novo expression of this molecule.

The MLL gene plays an important role in maintaining a cell type-specific HOX gene expression.24 HOX genes are essential for the controlled and regulated differentiation of blood cell progenitors into mature blood cells.25 Thus, the disruption of normal HOX gene expression patterns by a leukemogenic MLL fusion protein may be important for the development of either acute myeloid or lymphoblastic leukemia in progenitor cells with MLL rearrangements.10 Furthermore, it was shown that the MLL-LTG9 fusion protein inhibits the expression of several HOX genes.26 Therefore, the aberrant NG2 expression on acute leukemia cells could be due to dysregulated HOX gene expression as genes encoding adhesion molecules seem to be target genes for HOX proteins.272829

A possible explanation for the divergent moab 7.1 reactivity of acute leukemia cells with MLL rearrangements could be different NG2 expression patterns, which result from different translocations partners involved in 11q23 aberrations.3 More than 25 different genes are known to translocate to the MLL gene at the breakpoint cluster region at 11q23.10 The importance of the MLL fusion partners for leukemogenesis is highlighted by the finding that chimeric mice carrying the t(9;11) MLL-LTG9 fusion gene developed acute myeloid leukemia, but not mice with a truncated MLL gene.30 However, in our series we found no clear correlation between moab 7.1 reactivity and translocation partners: 28 of 31 (90%) cell samples with a t(4;11), 16 of 17 (94%) with a t(9;11), and three of five (60%) with a t(11;19) reacted with moab 7.1. Interestingly, cell samples of two AML with a t(3;11) and a t(6;11), respectively, did not react with moab 7.1 despite MLL rearrangements. Only two of six (33%) cell samples with a del(11)(q23) and MLL rearrangements were positive for moab 7.1.

The immunophenotypic marker profiles of moab 7.1-positive ALL cell samples were almost restricted to the pro-B-ALL subtype (33 pro-B-ALL, one common-ALL, one pre-B-ALL). These results are in accordance with previously reported data in childhood ALL.2 Pro-B-ALL cases with MLL rearrangements were shown to coexpress more often CD65s and less often CD13 and CD33 than pro-B-ALL cases without MLL rearrangements.419 Our data confirm these observations (Table 1). We did not detect any immunophenotypical pattern which could reliably distinguish between moab 7.1-positive and -negative leukemic cell samples with MLL rearrangements. The significantly lower expression of the adhesion molecule CD54 (ICAM-1) in moab 7.1-positive pro-B-ALL with t(4;11) observed in our study compared to other pro-B-ALL cases might be of relevance for the higher incidence of hyperleukocytosis, organomegaly, and CNS involvement in this leukemia subtype,5 as low CD54 expression correlates with these clinical parameters in childhood ALL.31 Moreover, NG2 itself may contribute to extramedullary manifestations in acute leukemia. Increased NG2 expression was shown to enhance metastatic properties of melanoma cells, possibly by modulating binding events mediated through the adhesion molecules CD44 and α4β1 integrin.32

In AML, most moab 7.1-positive samples showed morphologically a FAB M4/5 subtype and a myelomonocytic-differentiated immunophenotype with high expression levels of differentiation markers (CD65s, CD64, CD36) as well as low expression levels of progenitor antigens (CD34, CD117), both well-known characteristics of AML with MLL rearrangements.3334 Most moab 7.1-positive AML also expressed the adhesion molecule CD56 (NCAM) (33 of 38 patients). Moreover, two of eight patients with the recently described new entity of CD7+ CD56+ myeloid/NK-precursor cell acute leukemias reacted with moab 7.1.14 Only five of 84 (6%) CD56neg. myelomonocytic-differentiated AML samples (as defined by CD14 and/or CD64 positivity) were positive for moab 7.1, but 30 of 70 (43%) CD56pos. myelomonocytic-differentiated AML cases reacted with moab 7.1 (P < 0.00001). In contrast, all moab 7.1-positive ALL cases were found to be negative for CD56. In AML, CD56 expression is most common in myelomonocytic subtypes (FAB M4/5)35 and the FAB M2 subtype with t(8;21).36 Interestingly, on neuronal cells, CD56 can bind to chondroitin sulfate proteoglycans of the cell matrix.37 NG2 is a member of the chondroitin sulfate proteoglycan family and is thought to be involved in cell adhesion and migration mechanisms.11 Therefore, the aberrant coexpression of CD56 and NG2 on AML cells may be connected during leukemogenesis. Interestingly, it was recently shown that CD56 expression in AML with t(8;21) is associated with significantly shorter complete remission duration and survival.38 Whether the coexpression of CD56 and NG2 has any pathophysiological impact remains to be determined.

As moab 7.1 reacted with leukemic cells of patients with ALL, AML, and CD7+ CD56 myeloid/natural killer cell precursor acute leukemias, these findings suggest that the aberrant NG2 expression occurs at an early step in leukemogenesis on an immature non-committed progenitor cell with the potential to differentiate into several distinct lineages, as generally accepted for acute leukemias with MLL rearrangements.5 Moreover, the CD56 coexpression in the majority of NG2-positive AML raises the question whether these leukemias share a common progenitor cell with CD7+ CD56+ myeloid/natural killer cell precursor acute leukemias. Therefore, moab 7.1 may help to further define this newly proposed leukemia entity.

In conclusion, moab 7.1 seems to be the first antibody which reliably detects a protein with an expression pattern highly related to a specific genetic abnormality in acute leukemia cells. In our opinion, however, moab 7.1 does not obviate the need for molecular testing, especially as moab 7.1 does not distinguish between the different translocation partners involved in MLL rearrangements. In future studies, multicolor flow cytometry using carefully constructed antibody panels should be performed in order to evaluate more precisely the specificity and sensitivity of aberrant immunophenotypic features for the identification of acute leukemias carrying MLL rearrangements. Furthermore, we suggest that moab 7.1 should be evaluated as a promising tool for ‘minimal residual disease’ (MRD) detection by flow cytometry. Based on our findings, we recommend the integratation of moab 7.1 in the first screen antibody panel for immunophenotyping of all precursor B cell leukemias, especially pro-B-ALL, as well as for myelomonocytic-differentiated AML cases. Our ongoing prospective study will show whether moab 7.1 positivity without MLL rearrangements will define a subgroup of AML cases with different biological and clinical features.

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Acknowledgements

We would like to thank M Martin, K Liebezeit and K Ganzel for their excellent technical assistance and Dr A Schönberger (Beckman Coulter) for kindly providing the moab 7.1 for our investigations. The cell samples included in this study were sent from various hospitals in Germany participating in the ongoing ALL-BFM (coordinators: M Schrappe and H Riehm, Hannover), GMALL (coordinator: D Hoelzer, Frankfurt), AML-BFM (coordinator: U Creutzig, Hannover) and AML-CG (coordinators: T Büchner, Münster; W Hiddemann, München; B Wörmann, Braunschweig; W Berdel, Münster) trials. We thank the coordinators of the above studies for their continuous support as well as all clinicians providing cell samples for our investigations. This work was supported by grants of the ‘Deutsche José Carreras Leukämie Stiftung’ (JCLS 1998/NAT-3 to CW) and the ‘Deutsche Leukämie-Forschungshilfe’ (DLFH-98.04 to VR).

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Correspondence to C Wuchter.

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Wuchter, C., Harbott, J., Schoch, C. et al. Detection of acute leukemia cells with mixed lineage leukemia (MLL) gene rearrangements by flow cytometry using monoclonal antibody 7.1. Leukemia 14, 1232–1238 (2000) doi:10.1038/sj.leu.2401840

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Keywords

  • acute leukemia
  • MLL rearrangements
  • flow cytometry
  • moab 7.1

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