Acute Leukemias

CD2-positive B-cell precursor acute lymphoblastic leukemia with an early switch to the monocytic lineage


Switches from the lymphoid to myeloid lineage during B-cell precursor acute lymphoblastic leukemia (BCP-ALL) treatment are considered rare and thus far have been detected in MLL-rearranged leukemia. Here, we describe a novel BCP-ALL subset, switching BCP-ALL or swALL, which demonstrated monocytosis early during treatment. Despite their monocytic phenotype, ‘monocytoids’ share immunoreceptor gene rearrangements with leukemic B lymphoblasts. All swALLs demonstrated BCP-ALL with CD2 positivity and no MLL alterations, and the proportion of swALLs cases among BCP-ALLs was unexpectedly high (4%). The upregulation of CEBPα and demethylation of the CEBPA gene were significant in blasts at diagnosis, prior to the time when most of the switching occurs. Intermediate stages between CD14negCD19posCD34pos B lymphoblasts and CD14posCD19negCD34neg ‘monocytoids’ were detected, and changes in the expression of PAX5, PU1, M-CSFR, GM-CSFR and other genes accompanied the switch. Alterations in the Ikaros and ERG genes were more frequent in swALL patients; however, both were altered in only a minority of swALLs. Moreover, switching could be recapitulated in vitro and in mouse xenografts. Although children with swALL respond slowly to initial therapy, risk-based ALL therapy appears the treatment of choice for swALL. SwALL shows that transdifferentiating into monocytic lineage is specifically associated with CEBPα changes and CD2 expression.


Most patients diagnosed with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) never experience immunophenotypic changes that call the original diagnosis into question, and lineage switching to the myeloid lineage rarely occurs prior to complete remission. Thus far, case report findings suggest that MLL gene rearrangement represents a recurring genetic alteration.1 The MLL gene, which associates with various forms of lineage plasticity,1, 2, 3, 4, 5 encodes a transcriptional regulator of hematopoiesis and intrauterine development, and MLL fusions with various partner genes result in leukemias from different primary lineages. The differentiation plasticity of MLL-rearranged leukemias may already be visible at the time of diagnosis.6, 7 Another known example of lineage switching, mostly from myeloid to lymphoid phenotype, rarely occurs in BCR-ABLpos leukemias (ALL or CML blast crisis),8 and few epidemiological prospectively collected data have been published.5 Significant change of the phenotype during early phase of the treatment is generally a warning sign and questions about the optimal treatment strategy rise frequently.

The ALL-IC-BFM-2002 protocol includes molecular genetic and flow cytometry (FC) analyses of the bone marrow (BM) and peripheral blood (PB) at therapeutic days 8, 15 and 33.9 We identified two patients with CD2posBCP-ALL and atypical monocytosis in the PB and BM. This monocytosis appeared after treatment had begun, as revealed by morphology and FC, and rearrangements of the MLL gene were absent.

We first evaluated whether CD2pos BCP-ALL with monocytosis constituted a subset of ALL. The hypothesized that ALL subtype (switching BCP-ALL, swALL) was defined as BCP-ALL and was not classified as bilineal leukemia at diagnosis; rather, this subtype demonstrated a significant elevation in monocyte-like cells (monocytoids) derived from the leukemic clone in the BM or PB between days 1 and 33 of treatment. The leukemic nature of monocytoids was supported by the existence of cell population(s) at the intermediate B lymphoblast to monocytic stages (intermediate B/monocytoids) with distinct expression of CD14, CD19, CD33 and CD34.

Models of human hematopoiesis assume either early separation of the myeloid and lymphoid lineages, common lymphoid-myeloid progenitors or, more recently, sequential branching of specialized cells (erythroid, B and T lineages) from a backbone of ‘prototypic’ myeloid cells.10 In addition, a bipotential B-macrophage progenitor was identified in the human BM.11 Phylogenetically, B cells likely evolved from ancestral phagocytic cells,12 and T-cell receptor (TCR)δ rearrangements occurring early in lymphogenesis can be found in lymphocyte subsets but not in monocytes or granulocytes, indicating that the lymphoid/myeloid decision precedes the B/T/NK decision in a majority of cells.13, 14

Another example of lineage ambiguity is mixed phenotype AL,15 in which leukemic cells already express molecules of various lineages at diagnosis. Again, their low frequency indicates that ambiguity is not a general feature of lymphoblasts. Various B-cell malignancies including BCP-ALL may also evolve into histiocytic sarcoma or Langerhans histiocytosis.16, 17 The simultaneous presence of B cell and histiocytic clones has been described in B-cell lymphoma subtypes,18, 19 and both malignancies typically share identical Ig-TCR rearrangements.17, 20 These cases highlight the ability of malignant B-cells to transdifferentiate.

This study prospectively sought to determine the immunophenotypic features of swALL and investigate the nature of monocytoids in swALL. We also investigated the incidence, molecular genetics, underlying mechanisms and prognosis of swALL patients.

Patients and methods

For complete methodological details, see the Supplementary data.

Patients and therapy

In total, 708 Czech patients included in this study were diagnosed with centrally-evaluated primary BCP-ALL immunophenotype and treated according to the following protocols:ALL-BFM95 (09/1996–10/2002, n=269, including two patients who died at day of Dx), ALL-IC-BFM2002 (11/2002–10/2007 n=257), ALL-BFM2000 (11/2007–05/2010, n=154). Infants were treated according to the following protocols: POG9407 (n=5), Interfant1999 (n=12, including one patient who died at day of Dx) and Interfant 2006 (n=11). The patients (n=3) who died at day of Dx were excluded from the analysis of swALL incidence, but were included in the analysis of CD2 expression and its prognostic impact. Additional three swALL cases were included for molecular studies after 05/2010.

A validation cohort consisted of 44 patients newly diagnosed with BCP-ALL between 10/2010 and 07/2011 from Austria. The CD2pos Austrian patients were analyzed using a mAb combination including CD19, CD10, CD34 and CD14 at days 15 and 33. PB (days 5, 8 and 15) and BM (day 15) from patients with CD2pos BCP-ALL diagnosed in Zurich, Switzerland were analyzed using an eight-color swALL cytometry (8CSAC: CD45/CD14/CD10/CD20/CD19/CD34/CD33/CD3). This study was approved by the institutional review board and informed consent was obtained from patients and their guardians in accordance with the Declaration of Helsinki.


All patients in this study fit the definition of BCP-ALL.15, 21 An 8CSAC was used to assess the incidence of immunophenotypic changes between B lymphoid and monocytic lineages during treatment. This combination was analyzed in all the Czech BCP-ALL patients with available material at diagnosis and days 8, 15 and 33 between September 2007 and May 2010 (n=179). More details in supplementary information.

Sorting experiments and gating strategy

Sorting experiments were performed using the 8CSAC combination. Monocytoid cells were identified as CD14posCD33posCD34negCD19neg cells (Supplementary Figure 1). Intermediate B/monocytoids (CD14posCD19posCD33posCD34pos) were sorted in several samples.

Minimal residual disease (MRD)

The level of MRD was detected using 3-to 8-color FC as previously described,22, 23 (qPCR) targeted against patient-specific immunoglobulins (Igs) and TCR rearrangements was performed as previously described for samples and sorted subpopulations.24, 25

In vitro simulation of the first week of the therapy

Leukemic cells at diagnosis were collected from 12 patients (four with swALL and eight patients with other BCP-ALL subtypes) and isolated by Ficoll gradient. Unsorted cells were cultured for 4–8 days in the presence of 0, 5, 10 or 100 μg/ml prednisolone using RPMI supplemented with 10% heat-inactivated fetal bovine serum and penicillin-streptomycin. These prednisolone concentrations spanned plasma concentrations present during the anti-leukemic therapy26 and those leading to changes in B-cell marker expression.27 Immunophenotyping was performed every 24 h using 8CSAC.

Gene expression analysis

CEBPA gene expression was evaluated by quantitative RT-PCR in unsorted diagnostic samples. Physiological cell populations were sorted from PB control samples into the following populations: monocytes (CD14pos), granulocytes (CD45dimSide-scatterhigh) and B- (CD19pos) and T-lymphocytes (CD3pos). GMCSF-R and MCSF-R expression was evaluated by quantitative RT-PCR in sorted and unsorted samples and by FC in unsorted samples. For details, see the Supplements.

Multiplex PCR of 90 genes involved in lymphoid and myeloid development

Gene expression was analyzed on FC-sorted subpopulations (Supplementary Table 3). RT-PCR was performed in duplicate using TaqMan gene expression assays and TaqMan Universal Master Mix II with an ABI PRISM 7500 Fast RT-PCR System (Applied Biosystems, Foster City, CA, USA). The Delta Ct method was used to calculate the relative mRNA expression in relation to internal control genes (GUSB, HPRT1) selected using the NormFinder tool.28 MultiExperiment Viewer was used to perform hierarchical clustering, principal component analysis and comparison analyses.

Methylation of the CEBPA promoter region

The methylation status of diagnostic samples (16 swALL, 30 BCP-ALL, four T-ALL and 14 AML) was analyzed. More details in the Supplements.

Other genetic techniques

Single nucleotide polymorphism (SNP), IKAROS (IKZF1) and ERG alterations, cloning, sequencing, fusion gene determination and cytogenetics are described in the Supplements.

Xenotransplantation model

Thawed, viable unsorted cells obtained on diagnosis of swALL were intrafemorally transplanted into the NOD. Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NOD-scid IL2Rγnull, NSG) mouse strain. Cell viability was assessed by trypan blue staining. Engraftment was analyzed by FC every 2–48 weeks (details in supplementary information). Mice were intraperitoneally treated every 24 h with prednisolone at 30 mg/kg/day or with vehicle alone. The rate of engraftment was compared with our previously published cohort.29


Differences in continuous variables were analyzed with the Mann–Whitney U test unless otherwise stated. Fisher’s exact test was used to compare frequencies in 2 × 2 tables.


Characteristics of swALL patients

In total, 18 patients with swALL were identified (Table 1). BCP-ALL cases that were not classified as bilineal leukemia at diagnosis but revealed leukemia-derived monocytoid cells in the BM or PB in one or more samples between days 1 and 33 of ALL treatment were considered swALL patients. The relatedness between monocytoids and classical B lymphoblasts was demonstrated by the presence of a distinct B-to-monocytoid differentiation, which involved intermediate B/monocytoids (Figure 1, Supplementary Figure 1). Although the swALL patients were treated by different treatment protocols the initial phase until day 33 was always derived from the identical BFM corticosteroid-containing induction.

Table 1 Characteristics of patients with swALL
Figure 1

Dynamic changes in swALL. (a) The expression of CD19 and CD14 in BM specimens from a patient with swALL. Cells with low side scatter after the exclusion of debris and T cells are shown. Whereas at diagnosis (dx) the expression of CD19 dominated and only a few CD14pos events were observed at day 8 (d8), the CD14pos subpopulation was observed until day 15 (d15). (b) Monocyte-like morphology was observed for some cells at day 8 (ii) but not at diagnosis (i). (c) The percentage of CD14pos cells at standard time points until day 33 (d33) in swALL (filled symbols) and non-switching BCP-ALL patients (open symbols). The boxes range from the 25th to 75th percentiles with a line at the median, the whiskers indicate the 10th and 90th percentiles, and outlying values are indicated as individual circles. (d) The PB monocyte percentage in 15 swALL patients (146 values) is shown in comparison to the values observed in 70 control patients (non-switch BCP-ALL patients, 486 values). Each line connects the values of an individual swALL patient, and the maximum for each patient is highlighted by an oval and labeled with a number corresponding to the swALL unique patient number (UPN). The gray area represents the 10th to 90th percentile for the controls, and the dark gray area represents the 25th to 75th percentiles for the controls.

Unexpectedly high incidence of swALL

The incidence of swALL was estimated from a cohort of prospectively screened BCP-ALL cases using 8CSAC (09/2007–05/2010). Among the 179 unselected, prospectively evaluated primary BCP-ALL patients, seven (3.9%) were classified as swALL. Therefore, we may have missed several patients in a retrospective search for swALL patients among those who were CD2(partly)pos and/or had a poor prednisone response as this revealed only five patients among the 526 BCP-ALL patients (0.95%) diagnosed in the period between 09/1996 and 08/2007. Prospectively, the swALL patients comprised 7 of 16 (44%) CD2(partly)pos BCP-ALL patients but were absent among the CD2neg BCP-ALL patients (0 of 163; P<0.0001). In a prospective search among Austrian BCP-ALL patients, we found 2/44 cases (both with CD2pos BCP-ALL) with swALL (Table 1). In a Zurich patient with CD2pos BCP-ALL, the 8CSAC combination identified swALL features at days 8 (PB) and 15 (BM).

Increased monocytoid cells in swALL compared with other BCP-ALL patients

The frequency of BM and PB monocytoid cells increased within the first weeks of swALL treatment, exceeding 25% in some cases (Figure 1). The median peak of monocytoid cell frequency between treatment days 0 and 33 (time points as in Figure 1c) was 9.7% in swALL compared with 2.2% for other BCP-ALL cases (90th percentiles: 60% and 6.6%, respectively; P<0.0001). Although the dynamics differed among individual swALL patients, the most pronounced differences occurred at days 8 and 15. There were also smaller but significant differences in the BM at diagnosis and day 33. The level of intermediate B/monocytoid cells was significantly higher in swALLs or who were CD2pos at days 8 and 15 compared with other patients, but there was no difference at day 33 (Supplementary Figure 2).

Ig/TCR gene rearrangements in sorted monocytoid cells

Each swALL case demonstrated at least one clonal, specific Ig/TCR rearrangement at diagnosis, which enabled subsequent monitoring. In total, 46 specimens from diagnosis and days 8, 15 and 33 were available for sorting. In swALL, patient-specific Ig/TCR rearrangements were present in monocytoids from 26/31 (84%) MRDpos specimens (Figure 2, Supplementary Table 1 for specific Ig-TCR target results). In addition, six of eight BM specimens taken at diagnosis demonstrated monocytoids with leukemic involvement. In contrast, sorted monocytoids in only 1 of 14 specimens (7.1%) from patients with CD2neg BCP-ALL contained patient-specific Ig/TCR rearrangements (P=0.0061). In seven patients (swALL01, swALL07, swALL10, swALL11, swALL16, swALL17 and swALL18), intermediate B/monocytoids were sorted at diagnosis on day 8 and/or day 15, and leukemic involvement was confirmed in all cases.

Figure 2

Patient-specific Ig/TCR rearrangements in monocytoid cells. Monocytoid cells (cells expressing CD14 and CD33 but without CD34 or bright CD19, as defined in the text) were sorted in the presence of detectable MRD. The presence of patient leukemia-specific Ig/TCR rearrangements in sorted monocytoid cells is shown as filled red rectangles, the absence of a specific signal in sorted monocytoids despite MRD in an unsorted specimen is shown as open rectangles, and a situation in which the MRD of an unsorted specimen was >1 log higher than the percentage of B lymphoblasts is indicated by a hatched square with a dashed line. Blue rectangles show sorted intermediate B/monocytoid cells with detectable Ig/TCR rearrangements. Uninformative results are indicated with a tick and represent one of the two situations: positive MRD in unsorted specimens both by Ig/TCR and B-lymphoblast FC or negative MRD combined with either no sorting or a negative result. All findings in one control patient ‘CD2neg non-swALL BCP-ALL 08’ were uniformative and are not shown. BM: bone marrow; PB: peripheral blood; Dx; diagnosis sorting strategy shown in Supplementary Figure 1. The only patient with clonally abnormal monocytoid cells outside the typical swALL (CD2neg non-swALL BCP-ALL 02) demonstrated a poor response to therapy, and no abnormalities were found by conventional cytogenetics or PCR. Her blasts revealed an unaltered IKZF1 gene copy number despite expression of the dominant negative isoform Ik6, which was similar to patient swALL03. The sorting experiment was performed on day 15 when the BCP-ALL blasts predominated (41%). Unlike in swALL cases, PCR and FC MRD gave concordant results in this case, which were also visible at later time points.

If material was unavailable for sorting, we analyzed unsorted specimens and searched for discrepancies between high leukemic involvement determined by Ig/TCR rearrangements and low frequencies of typical BCP-ALL cells by FC. Frequencies of leukemic cells higher than 0.1% using Ig/TCR combined with undetectable or low (<10-fold below the respective Ig/TCR level) BCP-ALL CD19pos cells suggested that these specimens contained leukemic cells within non-B lineages. All other combinations (low Ig/TCR levels or high levels by both methods) were considered uninformative. In total, 14 of 40 (35%) swALL specimens contained leukemic infiltration that could not be explained by the frequency of BCP-ALL cells, whereas none of the 29 control specimens contained such a discrepancy.

No common genetic aberrancy in swALL patients

All swALL patients were euploid according to the DNA index, and none had the MLL gene rearrangements, FLT3-ITD (not analyzed in swALL 13–15), BCR-ABL, E2A-PBX or TEL/AML1. The findings from the SNP arrays are summarized in Supplementary Table 2. The number of detected somatic copy-number aberrations in swALL patients was lower than that reported for a cohort of unselected patients with BCP-ALL,30 similar to AML.31 Four of 16 patients (Table 1) had deletions at 9bp including homo- or hemizygous CDKN2A deletions. This finding is comparable to the frequency observed in the unselected BCP-ALL patients (27 of 112, P>0.1, data not shown). Two patients had deletions overlapping the CCDC26 gene at 8q24.21 and one patient had an additional copy of this gene due to chromosome 8 amplification. The frequency of IKZF1 alterations was higher in swALL cases compared with a previously examined BCP-ALL patient cohort32 (5/15 vs 17/150, P=0.032). ERG deletions were found in four patients (Table 1). Neither SNP arrays nor cytogenetics showed any other common aberration.

Because one of the key regulators of the monocytic/macrophage lineage is CEBPα, we examined the expression of this gene in ALL, AML and swALL samples by qRT PCR (Figure 3). A multiplex assay of the genes listed in Supplementary Table 3 was performed to compare the swALL (including day 8 subpopulations) expression profile to that of AML, BCP-ALL, physiological B cells, T cells and monocyte precursors. Both PCA and a heatmap demonstrate the gene expression in 62 sorted samples (Figure 5 and Supplementary Figure 4, respectively). In sorted samples from swALL patients, B lymphoblasts resemble B lymphoblasts of other BCP-ALL, monocytoids resemble developing normal monocytes (and to a lesser extent also monocytic AML), whereas the intermediate subpopulation was in between monocytoids and B lymphoblasts.

Figure 3

CEBPA expression in sorted physiological blood cells compared with AML and ALL. Median values are shown as horizontal lines. swALL cases are represented as a separate group. The expression of CEBPA was generally low in control BCP-ALL samples (CD2neg and MLL germline) and was similar to the level observed in sorted lymphocytes. CEBPA expression in swALL was significantly higher than that in control BCP-ALL, but significantly lower than that in AML (P=0.0043 and 0.0013, respectively).

Hypomethylation of the CEBPA promoter in the majority of swALL cases

In total, 14/16 swALL cases revealed hypomethylation in the CEBPA promoter in comparison to other BCP-ALL cases (Figure 4; differed in mean methylation levels, P=0.0039); CEBPA promoter methylation in swALL patients resembled that found in AML (mean methylation levels, P>0.05). Within the genetically defined subsets of BCP-ALL and T-ALL, hypermethylation of CEBPA promoter methylation was observed, Supplementary Figure 3). This CEBPA promoter methylation was in contrast to the CEBPα expression levels in MLL-rearranged ALL (high expression despite high promoter methylation) and BCR/ABLpos ALL (low expression despite promoter hypomethylation, Supplementary Figure 3), which can be explained by different mechanisms of CEBPα regulation in BCR/ABLpos ALL cases.33 Upon genome-wide methylation analysis, global promoter hypermethylation was identified in MLL-rearranged ALL infants in comparison to other ALL cases.34

Figure 4

Methylation status of swALL in comparison to control BCP-ALL (no MLL gene rearrangement, BCR/ABL, TEL/AML1 or hyperdiploidy) and AML. Each circle represents 1 of the 24 CpG dinucleotides in the CEBPA promoter region in 5′ to 3′ order (left to right), as identified by bisulfite sequencing. The location of the analyzed region was −295 to −59 3 bp upstream of the transcription start site (TSS), and 5 to 17 CpG island colonies for each patient were sequenced and subsequently analyzed. The degree of methylation is shown in greyscale; the saturation corresponds to the frequency of colonies with methylation in a given dinucleotide. Data from representative patients are shown. Additional cases are shown in Supplementary Figure 3.

SwALL patients respond slowly to initial treatment

Compared with the BCP-ALL cohort (n=73) treated with the ALL-IC-BFM 2002 protocol, patients with swALL (n=15) were more frequently classified as MRDpos by PCR in the BM at day 33 (13/15 and 27/73 in swALL and others; P=0.0002) and week 12 (7/13 and 6/68, P=0.0870). Importantly, the MRD of six patients was at or above 10−3 (day 33) combined with any positivity at week 12, indicating a slow response to the treatment.35 Only 1 of 11 BM specimens positive by PCR (range at day 33, 0.01%–4.1%) was detected by B-lymphoblast oriented FC or 8CSAC. We next compared the BM and PB MRD levels in swALL and other BCP-ALL patients at days 8 (n=12 and n=83 in BM swALL and others, n=11 and n=86 in PB) and 15 (n=15 and n=79 in BM, n=9 and n=78 in PB). The MRD levels at day 8 were significantly higher in only the PB of swALL patients (P=0.0086). At day 15, swALL patients had a higher MRD than other BCP-ALL patients in the BM (P=0.003) and PB (P=0.0019) (Supplementary Figure 6). In total two events in two swALLs occured: in swALL05, who developed a ‘secondary’ AML, which was CD14pos CD2negCD19neg and in a swALL15, who relapsed as BCP-ALL CD14negCD2posCD19pos. Rearrangements in the ‘secondary’ AML and original ALL cells were identical, indicating clonal relatedness.

Greater immunophenotypic changes after prednisolone treatment in vitro

To recapitulate the transdifferentiation of swALL blasts in vitro, the diagnostic blasts from swALL (n=4) and other BCP-ALL (n=8) patients were cultured and influenced with prednisolone. We observed decreased CD19 and CD34 expression together with an increased CD33 and CD14 expression (Figure 6).

Immunophenotypic changes in a xenotransplant model following corticosteroid treatment

Leukemia cells from swALL cases (n=6) were transplanted into NSG mice using an orthotopic intrafemoral technique without conditioning.29 Engraftment in the BM and spleen was achieved in only two of six swALL patients (33%), whereas human leukemia cells were present only in the blood in two additional patients. The yield of ALL cells in the spleen and BM were low (107 and 5 × 106, respectively). Interestingly, both swALL cases that were engrafted in NGS mice were the only swALL cases demonstrating a methylated CEBPA promoter. In one case, engrafted mice were treated with prednisone or vehicle to model anti-leukemia induction therapy. At days 8 and 15 of treatment, the immunophenotype partially shifted toward the monocytoid lineage (decrease of CD34 and increase of CD33 but no changes in CD19 and CD14 expression, Supplementary Figure 7) in mice treated with prednisolone but not in vehicle-treated controls. Interestingly, at day 8 of prednisolone treatment, the CEBPA promoter was highly demethylated compared with cells from vehicle-treated animals (Supplementary Figure 7).


Among the BCP-ALL patients, we identified 4–5% cases with monocytoid cells emerging during the induction treatment. These monocytoid cells had monocyte characteristics despite Ig/TCR rearrangements showing clonal relatedness to B lymphoblasts. Blasts with a switched lineage usually emerged transiently, and these swALL cases lacked previously known genetic markers for lineage ambiguity, namely the MLL gene rearrangements.

All observed swALL cases presented with CD2(partly)pos BCP-ALL. Our prospective search showed that 41% of patients with CD2(partly)pos BCP-ALL fulfilled the swALL definition, whereas no swALLs were found in CD2neg BCP-ALL cases. The percentage of intermediate B/monocytoids in CD2(partly)pos BCP-ALL cases was significantly higher at treatment day 8 in the BM compared with CD2neg BCP-ALL cases but significantly lower compared with swALL cases, indicating that some swALL cases may have been undiagnosed among patients with CD2(partly)pos BCP-ALL. CD2, thus appears to be associated with causal factors necessary but not sufficient for swALL features. The CD2 molecule is a member of the Ig-superfamily and, in humans, is mainly expressed on T and NK cells. On T cells, CD2 facilitates signaling from the TCR.36 Aberrant CD2 expression on B lymphoblasts is detected in fewer than 10% of the BCP-ALL patients.6, 37 We next asked whether swALL cases shared a common (epi-)genetic background. A ‘novel’ BCP-ALL subset was identified with a distinct gene expression profile,38 which was later shown to consist mostly of CD2pos cases with ERG gene deletions.39 Clappier and Zaliova et al.40, 41 observed an association between ERG deletions and CD2 positivity among the French and German BCP-ALL patients. Portion of the patients with ERG gene deletion also had monocytoid appearance of the blasts in morphology. We performed SNP arrays, MLPA and a breakpoint-specific PCR assay to reveal common copy-number variations. Although no genetic features were shared across the entire swALL cohort, an ERG gene deletion was found in 4/15 cases. IKZF1 alterations were more frequent among swALL patients than in the general BCP-ALL cohort. Ikaros is a key regulator of lymphoid lineage commitment, and it negatively regulates myeloid differentiation, including the inappropriate expression of myeloid genes.42 However, IKZF1 alterations cannot explain the evolution of swALL because IKZF1 appeared to be intact in a majority of swALL cases, and IKZF1 alterations were also present in non-swALL cases. Three swALL patients demonstrated alterations in the 8q24 locus (amplification or deletions) encompassing the RAM gene (retinoic acid-dependent modulator of myeloid differentiation, CCDC26). One previous study explored genome-wide alterations in childhood AML and identified copy-number gains, including those at the 8q24 locus, in 14% of patients.31 This locus was originally identified in a study investigating the downstream modulators of retinoic acid signaling,43 although the role of the RAM gene in lymphoid vs myeloid commitment has not yet been addressed.

Several models have demonstrated a role for enforced CEBPα expression in the transdifferentiation from B cells to macrophages.44, 45 We observed an increased CEBPA expression in swALL compared with the control BCP-ALL but lower expression in comparison to AML. In sorted intermediate B/monocytoid and monocytoid cells of swALL cases, we found gradually increased expression of PU.1 and the GM-CSF receptor and decreased expression of the B-lymphoid genes PAX5 and EBF-1, indicating a switch from the B to the myeloid program (Supplementary Figures 4, 5 and 8). Enforced expression of PU.1 together with CEBPα is required for promoting of macrophage differentiation in PU.1 inducible model.46 The expression of PU.1 and the GM-CSF receptor is directly driven by CEBPα,47, 48 and CEBPA was the only gene that was differentially expressed by lymphoblasts in swALL cases at diagnosis and before lineage switching; changes in other genes, including known differentiation regulators, occurred only during switching. Several mechanisms may be responsible for the increased expression of CEBPα in BCP-ALL. The CEBPA gene is involved in t(14;19)(q32;q13) chromosomal translocations in BCP-ALL,49 which leads to expression comparable to AML.50 However, this translocation was not identified in any of our swALL cases. Methylation of the CpG islands in the promoter region is important for the regulation of CEBPα expression,51 and a majority of swALL cases (14/16) demonstrated CEBPA promoter demethylation, which may explain the higher expression of CEBPA at diagnosis.

We next sought to establish a NSG mouse model to study this switching phenomenon in vivo, but durable engraftment was successful in only 2/6 swALL cases. This engraftment rate was lower than expected, as we achieved an engraftment rate70%29 using cells from non-swALL cases. Both engrafted swALL cases demonstrated CEBPA promoter methylation at diagnosis, which was preserved in engrafted cells. In mice treated with prednisolone, we observed an increased CD33 together with a decreased CD34 expression and demethylation of the CEBPA promoter in engrafted swALL blasts. We also observed changes in CD19 and CD14 in swALL patients, but not in mice.

Most (11/18) swALL patients had a high MRD level at day 33 (10−3), indicating a slow initial treatment response. Of these patients, the majority remained at least positive below 10−4 at week 12. Initial slower treatment response indicates a higher risk of relapse,35 however the real risk should be clarified on a prospective larger cohort of patients. Our experience indicates that risk-based ALL treatment (preferably based on MRD assessed by Ig/TCR rearrangements) is the treatment of choice for swALL children. However, in two swALL patients with the most prominent phenotypic change, we switched their treatment after day 33 to the Interfant-99 protocol with the aim of introducing combined myeloid-lymphoid therapy,52 and we later transplanted them from matched unrelated donors in CR1. In addition, the early development of morphologically and immunophenotypically unambiguous monocytic leukemia 6 months after cALL diagnosis in 1 patient (swALL05) raises caution for swALL prognosis, but the overall prognosis of swALL or CD2pos BCP-ALL was not worse compared with other BCP-ALL cases (data not shown). This result is in line with data from the 1990s describing a more favorable outcome for patients defined as CD19posCD2pos.37 This contrasts with the lineage switch in MLL-rearranged AL cases, which confers an extremely poor prognosis.5

SwALL diagnosis is also susceptible to false negative cytometric results. Indeed, FC could reliably identify only 1 of 11 PCR MRD-positive specimens taken at day 33 in our cohort of swALL patients. Contemporary B cell-oriented FC MRD fails when blasts lose B-lineage and/or progenitor markers such as CD19 and CD34. Thus far, we have not identified reliable immunophenotypic differences between switched monocytoids (that were positive by PCR, Supplementary Figure 1) and non-malignant monocytes. In addition, the expression profile of switched monocytoid cells resembles monocytes rather than monocytic leukemia cells (Figure 5, Supplementary Figure 4).

Figure 5

Principal component analysis of the expression of 90 genes in 62 sorted samples. Sorted cell subsets from swALL patients (B precursors prior switching at diagnosis, green, intermediate B/monocytoid cells at day 8, yellow, and monocytoids at day 8, red) are compared with reference leukemic cells of patients with either non-switching BCP-ALL or monocytic AML (both in dark gray) and to reference normal cells in various stages of development (B-cell precursors, T-cell precursors and developing monocytes, all in white). The reference cell populations were not distinguished by color, as all sorted normal cell populations clustered into three distinct regions; similarly both reference malignant populations clustered together with their non-malignant counterparts.

Figure 6

In vitro culture of freshly isolated blasts with and without prednisolone. (a) FC dot plots of blasts (I. - X.) from patient UPN swALL11 and a control case (UPN CD2neg non-swBCP-ALL3). Plots show cells following the exclusion of debris, events with a low forward scatter and T cells. (b) The percentage of CD14pos cells is shown for swALL patients (swALL11-gray, swALL02-red, swALL05- blue, swALL16-green) compared with CD2neg non-swBCP-ALL patients (n=8, black lines with triangles). Prednisolone-treated cells (5 μg/ml) are represented with filled symbols, and prednisolone untreated values are shown with open symbols. The percentage of CD14pos was significantly higher between days 1 and 4 in swALL samples compared with control patients. Monocytoid cells from patient swALL11 after cultivation in the presence of prednisolone at day 8 were sorted, and they contained the same leukemia-specific Ig/TCR rearrangements as the diagnostic sample.

One of the notable features of swALL is its switch dynamics. At the time of diagnosis, B lymphoblasts represent the most prevalent phenotype. The frequency of monocytoid cells at diagnosis is not statistically higher in swALL compared with other BCP-ALL cases. Monocytoid cells in swALLs at diagnosis ranged from 0.04–6.4%. The highest value was present in the swALL04, who received a dose of corticosteroids at a regional hospital before the diagnosis of ALL was made. The dose corresponded approximately to 2 days of treatment and we cannot exclude that a preceding therapy could modulate percentage of monocytes present at diagnosis. The frequency of monocytoid cells then increases early during treatment (Figure 1). The recapitulation of the change to a monocytic phenotype in vitro provided further evidence of the leukemic nature of these monocytoid cells. Probable factors enabling swALL to escape diagnostic detection include the lack of clear differences in the swALL phenotype at diagnosis, the temporary nature of the switch and the monocytic appearance of switched cells. Thus, swALL should be considered whenever high MRD is missed in BCP-ALL cases. One discrepancy between FC and Ig-TCR in a case with concurrent monocytosis was reported at day 19 by Neal et al.,53 and another patient without MLL aberrations but with a switch from BCP-ALL to CD14pos19pos monocytic leukemia carrying the same Ig-TCR rearrangement within 2 weeks of therapy was also described.54 Furthermore, Imataki et al. described a patient whose blasts switched within 2 months after diagnosis from BCP-ALL to monocytic leukemia with CD2 expression.55

Can the minority of monocytoid ALL-related cells detectable at diagnosis be the only source of subsequent monocytoid ALL-related cells? The gradual loss of CD19 and CD34 concurrent with an increase in CD14 and CD33 (Supplementary Figure 1) suggests that the continual change in lineage characteristics is the most likely explanation. Also the clonal relatedness between monocytoids and leukemic indicates that transdifferentiation is the most probable explanation of the phenomenon. Alternative explanation—that swALL represent a bilinear leukemia with slower clearance of monocytoid component during the treatment—is less probable. The in vitro data showing CD14pos cells even in cases with few or no detectable CD14pos cells at the beginning of culture also support transdifferentiation as an underlying mechanism. Different sensitivity of monocytoid cells to the initial therapy may also contribute to their relative increase during the therapy. Thus, swALL represents a subset of BCP-ALL in which significant quantities of monocytoid and intermediate B/monocytoid cells appear during the first month of treatment. For the first time, CD14posCD19neg cells with a monocytic morphology that are clonally related to leukemic B lymphoblasts were shown to exist as a distinct subset. The upregulation of CEBPα, which is related to CEBPA demethylation, appears to be an early event driving this switch, followed by changes in genes including PAX5, PU1, MCSFR and GMCSFR. The role of CD2 expression, which was positive in all observed swALL cases, remains to be elucidated. Although an increased proportion of patients with ERG or IKZF1 alterations was observed among the swALL cases, specificity and sensitivity of these changes indicate that they are not the driving alterations in swALL.


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This work was supported by grants: Sciex 09.043 (LS), NT 12397-4 (EF), GAUK 914613 (LS), GACR P301/10/1877 and NT13462 (EM). The FACS Aria instrument was supported by EU-Prague project CZ.2.16/3.1.00/24022, FWvD was supported by the Kay Kendall Leukaemia Fund and ZZ was supported by RVO-VFN64165/2012. This work was also supported by the project for conceptual development of research organization 00064203 and CZ.2.16/3.1.00/24022.We thank Alzbeta Vazna for sequencing; Iveta Janotova for data management; Veronika Kanderova for performence of the in vitro experiments; Pavel Semerak for technical assistance in sorting; Daniel Thurner, Angela Schumich and Daniela Morf for processing of FC samples; Katerina Muzikova for PCR analyses of sorted samples; and the Czech Pediatric Hematology Group for collaboration (Doctors Sterba, Timr, Mihal, Cerna, Prochazkova, Blazek and Hak) and for providing clinical information (Doctors. Timr, Smisek, Votava and Hak).

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Correspondence to E Mejstrikova.

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Author Contributions

LS analyzed the 8CSAC combination and performed in vitro and in vivo experiments, methylation analyses and MRD examinations; JS designed the analysis of methylation status, and was involved in the design of in vitro experiments and their interpretation and designed the expression studies; EF was responsible for the MRD examinations and expression studies; EV evaluated the morphology; JV performed the MLPA; MZ, FWvD and JZ performed the SNP arrays; LR performed a part of the expression studies, ZZ was responsible for the cytogenetics; KP designed and performed the expression array, GC performed the expression studies; MF interpreted the methylation studies; TK designed the sorting; KF analyzed the profiling data; JPB designed the in vivo experiments and performed the investigations of the Swiss patient; BB executed the xenograft model; MND performed the investigations of the Austrian patients; JT supervised the molecular genetics; JS managed the patients and contributed to data collection; OH wrote and reviewed the manuscript and EM discovered the key aspects of the switching phenomenon, identified the swALL patients, designed the research, analyzed the data and wrote the manuscript. All authors have read and approved the submission of the manuscript.

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Slamova, L., Starkova, J., Fronkova, E. et al. CD2-positive B-cell precursor acute lymphoblastic leukemia with an early switch to the monocytic lineage. Leukemia 28, 609–620 (2014).

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  • minimal residual disease
  • lineage switch
  • CCAAT/enhancer-binding protein alpha
  • acute lymphoblastic leukemia
  • Monocytes
  • CD2 Antigen

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