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Keywords:

  • paediatric acute lymphoblastic leukaemia;
  • CXCR4;
  • CXCL12;
  • BM-MSC;
  • Plerixafor

Summary

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Author contributions
  8. Disclosure of conflicts of interests
  9. References
  10. Supporting Information

Malignant cells infiltrating the bone marrow (BM) interfere with normal cellular behaviour of supporting cells, thereby creating a malignant niche. We found that CXCR4-receptor expression was increased in paediatric precursor B-cell acute lymphoblastic leukaemia (BCP-ALL) cells compared with normal mononuclear haematopoietic cells (< 0·0001). Furthermore, high CXCR4-expression correlated with an unfavourable outcome in BCP-ALL (5-year cumulative incidence of relapse ± standard error: 38·4% ± 6·9% in CXCR4-high versus 12% ± 4·6% in CXCR4-low expressing cases, < 0·0001). Interestingly, BM levels of the CXCR4-ligand (CXCL12) were 2·7-fold lower (= 0·005) in diagnostic BCP-ALL samples compared with non-leukaemic controls. Induction chemotherapy restored CXCL12 levels to normal. Blocking the CXCR4-receptor with Plerixafor showed that the lower CXCL12 serum levels at diagnosis could not be explained by consumption by the leukaemic cells, nor did we observe an altered CXCL12-production capacity of BM-mesenchymal stromal cells (BM-MSC) at this time-point. We rather observed that a very high density of leukaemic cells negatively affected CXCL12-production by the BM-MSC while stimulating the secretion levels of granulocyte colony-stimulating factor (G-CSF). These results suggest that highly proliferative leukaemic cells are able to down-regulate secretion of cytokines involved in homing (CXCL12), while simultaneously up-regulating those involved in haematopoietic mobilization (G-CSF). Therefore, interference with the CXCR4/CXCL12 axis may be an effective way to mobilize BCP-ALL cells.

A stem cell niche is a specific site in tissues where stem cells reside, undergo self-renewal and produce large numbers of progeny (differentiation). In bone marrow (BM) two niches have been identified: the osteoblastic (close to the bone) and the vascular niche (close to blood vessels) (Lilly et al, 2011; Park et al, 2012). Stromal cells, including reticular and mesenchymal cells, are common components of both niches. Functionally, these cells support haematopoietic stem cell (HSC) adhesion, quiescence, chemotaxis and, in the case of the vascular niche, differentiation (Lilly et al, 2011). Thus, HSC properties and functional responses depend on specific interaction with BM niches.

Defects in haematopoiesis in the BM can be due to dispersion of healthy cells by malignant cells but may also occur in the setting of relatively low tumour burden. Leukaemic blasts interact with the haematopoietic microenvironment in order to maintain self-renewal and to mitigate cytotoxic chemotherapy effects (Ishikawa et al, 2007; Lane et al, 2009; Nervi et al, 2009; Zeng et al, 2009). This environment-mediated drug resistance can be mediated by soluble factors, induced by cytokines, chemokines and growth factors (GF) secreted by tumour stroma; or via adhesion of tumour cell integrins to stromal cells or to components of the extracellular matrix (Meads et al, 2008). Increased sensitivity to chemotherapy after leukaemic blast mobilization indicates that disruption of these interactions may have therapeutic value (Meads et al, 2009).

Leukaemia stem cells (LSCs) and leukaemia propagating cells (LPCs) may find (physical) refuge within the BM niche during chemotherapy, and eventually contribute to disease relapse (Vormoor, 2009; Rehe et al, 2013). Interestingly, LSCs that infiltrate the BM (Sipkins et al, 2005; Ishikawa et al, 2007) interfere with the normal HSC-microenvironment homeostasis, creating a leukaemic BM niche (Colmone et al, 2008). It is well established that CXCR4-positive leukaemic cells migrate to specific CXCL12-positive vascular niches in the BM (Sipkins et al, 2005; Burger & Kipps, 2006). Here, we investigated the role of the leukaemic BM microenvironment and, specifically, the role of the CXCR4/CXCL12 axis in paediatric precursor B-cell acute lymphoblastic leukaemia (BCP-ALL). We showed that CXCR4 is highly expressed in paediatric BCP-ALL cells and that this correlated with an unfavourable clinical outcome. Interestingly, newly diagnosed BCP-ALL patients have significantly lower CXCL12 serum levels than BCP-ALL patients that received induction therapy and non-leukaemia control patients. Furthermore, we showed that CXCL12 secretion levels were inversely correlated with the amount of leukaemic cells present in in vitro mesenchymal stromal cell (MSC) co-cultures, while granulocyte colony-stimulating factor (G-CSF) secretion levels are increased. We therefore suggest that current treatment protocols solely aimed at targeting leukaemic blasts may be expanded in targeting also interactions with their supporting neighbouring cells.

Methods

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Author contributions
  8. Disclosure of conflicts of interests
  9. References
  10. Supporting Information

Serum sample collection

Bone marrow and peripheral blood (PB) samples were collected from children with untreated newly diagnosed ALL (initial diagnosis ALL) and after completion of the first two courses of chemotherapy (protocol IA and IB; ALL in remission) of the Dutch Childhood Oncology Group (DCOG) ALL-10 protocol. First aspirates BM samples (pure without any blood contamination) were used for cytokine expression analysis. This study was approved by the Institutional Review Board, and informed consent was obtained before initial treatment from parents or guardians.

Isolation of BCP-ALL cells

Bone marrow and PB samples from children with newly diagnosed BCP-ALL, and from children with no haematological disorder (normal bone marrow; nBM), were processed as previously described (Den Boer et al, 2003). Informed consent was obtained from parents or guardians. Briefly, mononuclear cells were isolated by centrifugation over a Lymphoprep density gradient. Blast percentages were determined microscopically using a May-Grünwald Giemsa staining. If necessary, samples were enriched for leukaemic blasts to >90% by removing non-leukaemic cells using immunomagnetic beads.

Isolation and expansion of MSC from leukaemic patients

Bone marrow aspirates were collected from children with ALL at first diagnosis (before start of therapy), after two courses of chemotherapy and non-leukaemic controls. In order to isolate MSC, BM aspirates were cultured in MSC expansion medium consisting of Dulbecco's modified Eagle medium (DMEM) low glucose medium (Invitrogen, Life Technologies, Breda, the Netherlands), 15% fetal calf serum (FCS) (Integro, Zaandam, the Netherlands), fungizone (Invitrogen), gentamycin (Invitrogen), freshly supplemented with 10−4 mol/l ascorbic acid (Sigma, Zwijndrecht, the Netherlands) and 1 ng/ml basic fibroblast growth factor (bFGF; Serotec, Kidlington, UK). After 24 h, non-adherent cells were removed and adherent MSCs were further expanded in MSC expansion medium. After a maximum of 10 d, MSC colonies were washed once with PBS and harvested using Trypsin (Invitrogen). Cells were cultured for one or two additional passages before they were frozen in liquid nitrogen for future experiments.

Bone marrow-MSC were characterized by flow cytometry using a specific CD antibody panel [including haematopoietic (CD14, CD34, CD45) and mesenchymal markers (CD13, CD29, CD54, CD73, CD166)]. Furthermore, we confirmed that the BM-MSC isolates were able to differentiate towards the adipogenic (Oil red O staining), osteogenic (Alizarin red S staining) and chondrogenic lineage (Thionin and Collagen-II staining).

Reverse Phase Protein Array

Reverse Phase Protein Array (RPPA) was performed as described previously (Paweletz et al, 2001; Petricoin et al, 2007; Zuurbier et al, 2010) in collaboration with Dr. Emanuel Petricoin, George Mason University, USA. Briefly, leukaemic cells from children newly diagnosed with ALL and mononuclear bone marrow cells from non-leukaemic controls were lysed in Tissue Protein Extraction Reagent (TPER; Pierce Biotechnology, Rockford, IL, USA) supplemented with 300 mmol/l NaCl, 1 mmol/l orthovanadate and protease inhibitors. Cell lysates were spotted in triplicate twice (in total each sample was represented by six spots on each slide) on glass-backed nitrocellulose-coated array slides (FAST slides; Whatman Plc, Kent, UK) by three hits performed by the Aushon Biosystems 2470 arrayer (Aushon Biosystems, Billerica, MA, USA). The first slide in every series of 15 slides was used to determine the total protein amount by Sypro Ruby Protein Blot Stain (Invitrogen) staining followed by visualization on a NovaRay CCD fluorescent scanner (Alpha Innotech, San Leandro, CA, USA). The slides were stained with a CXCR4 antibody (ab2074; Abcam, Cambridge, UK) followed by incubation with a biotinylated secondary antibody (BA-1000; Vector Laboratories, Burlingame, CA, USA) using a DAKO cytomation autostainer. Slides were scanned using the NovaRay scanner. All slides were analysed with the MicroVigene v2.8.1.0. software (VigeneTech, Carlisle, MA, USA). Finally, CXCR4 levels were calculated relative to the total amount of protein per sample.

Extracellular CXCR4 FACS analysis

Acute lymphoblastic leukaemia cells were incubated with 1 or 10 μmol/l Plerixafor (AMD3100, A5602; Sigma) for 4 or 24 h. Cells were collected, washed with PBS + 2% fetal bovine serum (FBS), incubated with a phycoerythrin (PE)-labelled monoclonal anti-human CXCR4 (555974 (clone 12G5), BD Biosciences, San Jose, CA, USA) antibody for 30 min, washed with PBS + 2% FBS and analysed by flow cytometry (Accuri; BD Biosciences). PE-labelled isotype antibody (555574, BD Biosciences) served as an isotypic control. Cells that were incubated without AMD3100 were included as a positive control.

Collection of MSC conditioned medium (MSC-CM)

Conditioned medium of 16 MSC samples from initial diagnosis (untreated), 11 paired MSC samples after two courses of chemotherapy (day 79), and 9 MSC samples from non-leukaemic origin (normal) were collected. For this, subconfluent MSC were trypsinized and plated in T75 flasks at a concentration of 2·5 × 106 cells per flask. Cells were left to adhere for 24 h before medium was replaced with 10 ml MSC expansion medium. After 72 h, conditioned medium was collected, centrifuged to remove any cellular debris, aliquotted and stored at −80°C until further use.

Collection of MSC, ALL and MSC-ALL conditioned medium

Conditioned medium of MSC, ALL and co-cultures thereof were collected. Subconfluent MSC were trypsinized and plated at a density of 20 000/cm2. Cells were left to adhere for 24 h before RPMI medium containing various amounts of ALL cells were added to each well. In the Plerixafor experiments, leukaemic cells were pre-incubated with different concentrations of Plerixafor for one hour at 37°C. After 72 h, conditioned medium was collected, centrifuged to remove any cellular debris, aliquotted and stored at −80°C for cytokine measurements by means of enzyme-linked immunosorbent assay (ELISA) and/or Luminex.

Profiling of cytokine and chemokine expression patterns

The expression profile of secreted proteins in MSC-CM was measured using the Luminex fluorescent bead human cytokine and chemokine immunoassays (MILLIPLEX MAP; Millipore, Billerica, MA, USA) according to the manufacturer's instructions. Two standard-sensitivity pre-mixed multipanels were used for the simultaneous quantification of multiple human cytokines and chemokines, including CXCL12 and G-CSF. A pre-mixed standard dilution curve, containing a fixed concentration of each analyte, was used for quantitative analysis (Millipore). According to the manufacturers' protocol, two quality controls were included as positive controls and for inter-plate comparisons. Additionally, in vitro expression of CXCL12 was also analysed by specific duoset sandwich ELISA according to the manufacturers' instructions (DY350; R&D systems, Minneapolis, MN, USA).

Statistical analysis

Mann–Whitney U-Test was used to compare CXCR4 and CXCL12 protein expression levels between two different groups. The Wilcoxon signed-rank test was used when comparing paired patient samples. Event-free survival (EFS) rates were determined using Cox's univariate and multivariate proportional hazard analyses with non-response to induction therapy, disease relapse, the emergence of secondary malignancies and death in remission as events. The cumulative incidence of relapse (CIR) with death as a competing event was calculated as described by Fine and Gray (1999) considering non-response and relapse as an event. Multivariate analysis included CXCR4 protein expression, white blood cell (WBC) count, age at diagnosis, cytogenetic subtype and sample source as covariates. CIR was computed with the statistical environment R version i386 2.15.1 using Bioconductor packages (R Development Core Team, 2013). The other analyses were performed with spss Statistics version 20 (SPSS Inc., Chicago, IL, USA). All tests were two-tailed and a P-value <0·05 was considered significant.

Results

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Author contributions
  8. Disclosure of conflicts of interests
  9. References
  10. Supporting Information

High CXCR4 protein expression is correlated with a poor prognosis in paediatric ALL

The CXCR4/CXCL12 axis is not only important in normal haematopoiesis; it was also shown to facilitate homing and engraftment of BCP-ALL cells to the BM of non-obese diabetic severe combined immunodeficiency (NOD/SCID) mice (Shen et al, 2001). In this study, we observed a significant higher expression of CXCR4 protein of leukaemic cells taken at initial diagnosis of 151 precursor B-ALL patients compared with mononuclear cells obtained from 10 non-leukaemic control patients (< 0·0001; Fig 1A). Based on CXCR4 expression levels, we divided the 151 precursor BCP-ALL patients in three equal groups: low, intermediate and high expression. High CXCR4 expression correlated with unfavourable cytogenetic subtype, PB localization and high WBC count at diagnosis (Table SI and Figure S1). In addition, high CXCR4 protein expression at initial diagnosis was associated with a poor outcome in paediatric BCP-ALL patients (5-year CIR ± standard error: 38·4% ± 6·9% in CXCR4 high versus 12% ± 4·6% in CXCR4 low expressing patients, < 0·0001; Fig 1B). Univariate analysis revealed that high expression of CXCR4 at diagnosis, WBC count at diagnosis, cytogenetic subtype and source (BM or PB) were statistically predictive for clinical outcome (Table 1). In a multivariate model including the aforementioned factors, we showed that high expression of CXCR4 was the most independent prognostic factor in paediatric BCP-ALL (= 0·036) (Table 1). We also eliminated sample source as a covariate and performed the multivariate analysis on BM only (Table SII). This multivariate model showed that high expression of CXCR4 remained the most independent prognostic factor in paediatric BCP-ALL (= 0·035) (Table SII).

Table 1. Univariate and multivariate analysis of prognostic factors in paediatric ALL patients. Prognostic value of WBC count, age at diagnosis, cytogenetic subtype, sample source and CXCR4 protein expression levels was tested in a univariate and multivariate analysis in paediatric BCP-ALL patients. Statistical analysis of prognostic factors was performed by the Cox proportional hazard model, based on event-free survival. All variables were analysed as categorical variables. This analysis was fitted on 151 paediatric ALL patients from whom all variables were available. Hazards ratio depicted for CXCR4 expression and subtype are a result of comparing high versus low or unfavourable versus favourable, respectively. Cytogenetic subtypes: favourable, ETV6-RUNX1 and hyperdiploid; intermediate, TCF3-rearranged and B-others [BCP-ALL patients without an BCR-ABL1, ETV6-RUNX1, KMT2A (also known as MLL) translocation that are neither hyperdiploid or BCR-ABL1-like]; unfavourable, BCR-ABL1, BCR-ABL1-like and KMT2A-rearranged.
 UnivariateMultivariate
HR (95% CI)P-valueHR (95% CI)P-value
  1. HR, hazard ratio; 95% CI, 95% confidence interval; WBC, white blood cell. CXCR4 expression is relative, calculated as CXCR4 expression relative to the total amount of protein per sample.

  2. P-values indicated in bold are statistically significant (i.e. P < 0.05).

CXCR4 expression  0·003   0·036
Low (<19 500), n = 511 1 
Intermediate, n = 504·8 (2·0–11·9) 0·001 3·3 (1·3–8·5) 0·014
High (≥23 250), n = 503·9 (1·6–9·8) 0·004 2·2 (0·8–6·0)0·131
WBC count (×109/l)
<50, n = 821 1 
≥50, n = 692·1 (1·1–3·7) 0·015 1·1 (0·6–2·2)0·673
Age (years)
<10, n = 1081 1 
≥10, n = 431·5 (0·9–2·8)0·1531·2 (0·6–2·2)0·606
Cytogenetic subtype  0·009  0·332
Favourable, n = 471 1 
Intermediate, n = 432·0 (0·8–4·8)0·1251·5 (0·6–3·9)0·405
Unfavourable, n = 613·3 (1·5–7·3) 0·003 1·9 (0·8–4·6)0·146
Source
Peripheral blood, n = 491 1 
Bone marrow, n = 1020·3 (0·2–0·6) <0·0001 0·5 (0·3–1·0)0·063
image

Figure 1. CXCR4 protein expression is a poor prognostic factor in BCP-ALL. (A) Total CXCR4 protein expression levels in leukaemic cells of newly diagnosed paediatric precursor B-ALL patients (BCP-ALL) compared with that of normal mononuclear cells of non-leukaemic control patients (NORMAL) (Normalized values: CXCR4 levels were calculated relative to the total amount of protein per sample) (B) Cumulative incidence of relapse and non-response curve for CXCR4 protein expression in paediatric ALL patients with death as a competing event. CXCR4 expression was divided into three equal subgroups CXCR4 high (23 250–30 500), CXCR4 intermediate (19 500–23 249), CXCR4 low (12 323–19 499).

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CXCL12 serum levels in newly diagnosed patients are significantly lower than that of patients in morphological remission and non-leukaemic patients

The interaction of CXCL12 with its receptor (CXCR4) is important in retaining ALL cells in the BM (Bradstock et al, 2000; Shen et al, 2001). Together with the fact that CXCL12 expression was reported to be up-regulated in regions of hypoxia (which occurs if leukaemic cells become numerous) (Hitchon et al, 2002; Ceradini et al, 2004), we expected that CXCL12 protein levels would be elevated in the tumour niche. Surprisingly, we found 2·7-fold (= 0·005) and 1·5-fold (= 0·011) lower CXCL12 levels in BM- and PB-derived serum respectively, of 11 untreated ALL patients at the time of diagnosis compared to serum of seven non-leukaemic control patients (Fig 2). Moreover, PB and BM serum levels taken when patients were in remission after induction chemotherapy (day 79) were similar to the levels seen in non-leukaemic patients (Fig 2). As BM stromal cells are able to produce and secrete CXCL12, we next isolated MSC from newly diagnosed paediatric BCP-ALL patients (untreated) and compared the secreted levels of CXCL12 to that of samples taken when patients were in morphological remission (day 79) and non-leukaemic controls (normal). We showed that all MSC isolates were capable of secreting CXCL12 and that there was no significant difference in secreted levels between MSC isolated from ALL cases at diagnosis, ALL cases in remission at day 79 and non-leukaemic samples (Fig 3). This indicates that the reduced serum levels of CXCL12 in untreated newly diagnosed patients were not caused by the reduced capacity of MSC cells to produce CXCL12.

image

Figure 2. CXCL12 serum levels in leukaemic patients and controls. (A) bone marrow (BM) and (B) peripheral blood (PB) serum samples were collected from 11 children at initial diagnosis of ALL (untreated) and at remission after two course of chemotherapy (day 79) of the DCOG ALL-10 protocol. CXCL12 serum expression levels were determined and compared with BM (A) and PB (B) serum levels of seven non-leukaemic patients (normal) using the Luminex platform.

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image

Figure 3. CXCL12 production by MSC. Mesenchymal stromal cells (MSC) were isolated from untreated newly diagnosed ALL patients (untreated), paediatric ALL patients in remission after having received chemotherapy (day 79), and non-leukaemic controls (normal). After 72 h, conditioned medium was collected and CXCL12 secretion levels were determined using the Luminex platform.

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Elevated CXCL12 levels in BM versus PB compartment create a gradient

Given that CXCR4-positive cells home from the PB towards the BM, a gradient in CXCL12 levels between the BM (high) and PB (low) compartment is expected. Indeed, CXCL12 levels were twofold higher in BM-derived serum compared to PB-derived serum in matched cases, both in samples taken after chemotherapy (ALL in remission) (= 0·001) and those of non-leukaemic patients (= 0·016; Fig 4). As CXCL12 serum levels in untreated patients were already low at time of diagnosis, no gradient was observed in these samples.

image

Figure 4. Gradient in serum levels of CXCL12 from BM to PB compartment. CXCL12 secretion levels of matched bone marrow (BM) and peripheral blood (PB) serum samples were compared in 11 children at (A) initial diagnosis or (B) after two courses of chemotherapy when in remission and (C) seven non-leukaemic controls.

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Decrease in CXCL12 levels is not due to consumption by the leukaemic cells

The lower CXCL12 serum levels at the time of diagnosis might be explained by consumption of CXCL12 by the high number of leukaemic cells in the bone marrow niche. To investigate this, we co-cultured MSCs with increasing numbers of leukaemic cells and measured the secreted levels of CXCL12. In three independent experiments we observed that the levels of secreted CXCL12 inversely correlated with the number of leukaemic cells (Fig 5; blue and black lines). In order to investigate whether this decrease was caused by CXCL12 uptake by the leukaemic cells, we added the CXCR4 antagonist Plerixafor (AMD3100). Interaction of Plerixafor with CXCR4 prevents CXCL12 from binding and also blocks the binding of CXCR4 antibody (12G5) to the receptor (De Clercq, 2000; Juarez et al, 2003). As shown in Figure S2 (left-hand panel), Plerixafor bound to the CXCR4 receptor thereby decreasing antibody-staining levels. However, blockage of CXCR4 by Plerixafor did not alter CXCL12 levels in the medium (Fig 5; blue and red lines) indicating that the lower levels of CXCL12 at the time of diagnosis cannot be explained by consumption by leukaemic cells. These low levels at the time of diagnosis therefore need to be caused by a negative feedback-loop on CXCL12 production triggered by the leukaemic cells.

image

Figure 5. CXCL12 secretion levels in single and co-cultures of MSC with leukaemic cells. In three independent experiments [(A) Experiment1, (B) Experiment2 (C) Experiment3], secretion of CXCL12 in the supernatant of ALL cells (black lines) was compared with the secretion levels in MSC+ALL co-cultures (blue lines). A variable amount of leukaemic cells (depicted on the x-axis) was cultured in the presence or absence of a fixed amount of MSC for 72 hours. CXCL12 levels in the supernatant of these cultures were determined by sandwich ELISA. In addition, co-culture experiments of MSC and ALL cells were performed in the presence of Plerixafor (red lines).

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Decreased CXCL12 levels inversely correlate with increased levels of G-CSF

Given that the CXCR4 and G-CSF pathways seem to overlap (Kim et al, 2006), we measured secreted G-CSF levels in our co-culture experiments. Although CXCL12 levels decreased when leukaemic cells numbers increased in these co-cultures, we observed a clear up-regulation for G-CSF (Fig 6). Although G-CSF is already produced by leukaemic and stromal cells, expression levels were remarkably induced when these cells were cultured simultaneously. In addition, we also found elevated G-CSF levels in untreated paediatric BCP-ALL serum samples compared to healthy individuals, both in BM-derived samples as well as in PB-derived samples (Figure 3).

image

Figure 6. CXCL12 and G-CSF secretion levels in co-culture experiments of MSC and leukaemic cells. Various amounts of leukaemic cells were cultured in the presence (grey bars) or absence (black bars) of a fixed amount of mesenchymal stromal cells (MSC) cells. Supernatant was collected after 72 h of culture and the levels of (A) CXCL12 and (B) granulocyte colony-stimulating factor (G-CSF) were determined using the Luminex platform.

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Discussion

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Author contributions
  8. Disclosure of conflicts of interests
  9. References
  10. Supporting Information

The CXCR4-CXCL12 axis is involved in many different types of cancer (Teicher & Fricker, 2010). Regarding haematological malignancies, CXCR4 is best studied in patients with acute myeloid leukaemia (AML). In this group of patients, overexpression of CXCR4 is an independent predictor of a poor prognosis (Rombouts et al, 2004; Dommange et al, 2006; Spoo et al, 2007; Tavernier-Tardy et al, 2009). Little is known regarding CXCR4 expression in BCP-ALL patients. Crazzolara et al (2001) demonstrated that paediatric B-ALL cases with high CXCR4 protein expression had significantly more prominent leukaemic cell infiltration of the liver or spleen compared with cases that had low CXCR4 expression. However, Schneider et al (2002) analysed a small cohort of paediatric BCP-ALL patients and could not confirm these observations, although they did report a shortened disease-free survival in patients with high CXCR4 expression. Our data supports the conclusion that high CXCR4 protein expression is correlated with an increased risk of relapse and poor outcome in paediatric BCP-ALL patients. In addition, we showed that this is independent of WBC count, age at diagnosis, cytogenetic subtype and sample source. Interestingly, CXCR4 expression levels were found to be significantly higher in samples isolated from the PB compartment compared to samples isolated from the BM compartment (Table SI and Figure S1). This might be explained by the fact that BCP-ALL cells localized in the BM compartment no longer need CXCR4 and/or that CXCR4 is internalized upon CXCL12 binding.

Thus far, reports regarding CXCL12 serum levels in leukaemic patients are limited. Khandany et al (2012) reported that CXCL12 serum levels were significantly increased in adult ALL patients compared with controls. On the other hand, Colmone et al (2008) found decreased CXCL12 levels in the serum of NALM6-engrafted (SCID) mice (Colmone et al, 2008). In the present study, we found that, in ALL patients, CXCL12 serum levels were significantly lower at diagnosis compared to remission after chemotherapy (day 79) and non-leukaemic control patients. This lower serum level could not be explained by consumption of CXCL12 by the leukaemic cells or by an altered production capacity of MSC at the time of diagnosis. In contrast, we observed that leukaemic cells negatively affected CXCL12 production by the MSC. We checked whether the variance in secreted levels correlated to MRD levels at the end of induction (ALL in remission). However, MRD status did not correlate with CXCL12 expression levels in these patients (Figure S4). Of interest, we observed that a drop in CXCL12 production coincided with an elevated level of G-CSF when leukaemic cell numbers increased. G-CSF has been shown to mobilize HSC and myeloid cells from the BM into the blood compartment by down-regulating CXCR4 expression and attenuating their responsiveness to CXCL12 (Kim et al, 2006). Hypothetically, the increase in G-CSF and concomitant drop in CXCL12 production observed in the present study may trigger the release of lymphatic leukaemic cells from a crowded BM into the blood compartment and may foster the spreading of the disease. These circulating leukaemic cells can be targeted with traditional chemotherapeutic drugs. Adult patients with relapsed or refractory AML treated with Plerixafor in combination with low-dose cytarabine, aclarubicin and G-CSF had higher response rates than those without Plerixafor (Saito et al, 2000; Lowenberg et al, 2003; Qian et al, 2007; Wei et al, 2011). Interestingly, combination of G-CSF with Plerixafor increases the percentage of persons that mobilize sufficient stem cells for transplantation compared to G-CSF alone (Anonymous, 2007). Plerixafor is now approved by the US Food and Drug Administration in combination with G-CSF to mobilize HSC to the PB for collection and subsequent autologous transplantation in patients with non-Hodgkin lymphoma and multiple myeloma (DiPersio et al, 2009a,b). We observed that CXCL12 levels were low at the time of diagnosis, when leukaemic cells were numerous in the bone marrow, and increased upon chemotherapy treatment to levels seen in non-leukaemic controls. This, together with the observation that these lower levels seem to be caused by leukaemic cells, suggests that interference with the CXCR4/CXCL12 axis may be an effective way to mobilize ALL cells; the more ALL cells become mobilized, the less ALL cells may escape from combination chemotherapy. Promising results have already been obtained in a paediatric BCP-ALL mouse model, in which NOD/SCID mice were transplanted with newly diagnosed primary patients cells. Prolonged exposure to small molecule CXCR4 antagonists (including Plerixafor) reduced the number of leukaemic cells in the spleen and PB, and significantly reduced their dissemination to extramedullary sites (Juarez et al, 2007; Welschinger et al, 2013). As a next step, clinical trials using AMD3100 (in combination with other mobilizing agents such as G-CSF) to sensitize BCP-ALL cells to chemotherapy need to be conducted to determine whether such a strategy could improve responses to chemotherapy and patient outcomes.

Acknowledgements

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Author contributions
  8. Disclosure of conflicts of interests
  9. References
  10. Supporting Information

The authors would like to thank Valerie Calvert and Emanuel Petricoin at the Centre for Applied Proteomics and Molecular Medicine at the George Mason University, Manassas, USA, for performance of the reverse phase protein arrays. This project was funded by the Dutch Cancer Society grants EMCR 2007-3718 (MLDB, RP) and EMCR 2010-4687 (MLDB) and the Sophia Foundation for Medical Research grant SSWO-658 (AVDV, MLDB).

Author contributions

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Author contributions
  8. Disclosure of conflicts of interests
  9. References
  10. Supporting Information

LCJVDB, AVDV, RP and MLDB were involved in study design, experimental execution, data interpretation and writing of the manuscript. MEW, MJGAT and MWL contributed to experimental procedures. WHT and IMVDS contributed to serum sample collection and correction of the manuscript. MLDB was principal investigator. In addition, all authors contributed to and approved the final manuscript.

References

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Author contributions
  8. Disclosure of conflicts of interests
  9. References
  10. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Author contributions
  8. Disclosure of conflicts of interests
  9. References
  10. Supporting Information
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bjh12883-sup-0001-SupplementalData.docWord document616K

Data S1. Disturbed CXCR4/CXCL12 axis in paediatric precursor B-cell acute lymphoblastic leukaemia.

Fig S1. Correlation between CXCR4 protein expression and co-variates of the multivariate analysis.

Fig S2. Plerixafor binds to the CXCR4 receptor on leukaemic cells.

Fig S3. G-CSF serum levels in leukaemic patients and controls.

Fig S4. CXCL12 serum levels in MRD-negative and -positive ALL patients.

Table SI. Distribution of poor prognostic factors in the three CXCR4-expressing groups.

Table SII. Prognostic value of WBC count, age at diagnosis, cytogenetic subtype, and CXCR4 protein expression levels was tested in a univariate and multivariate analysis in paediatric BCP-ALL patients.

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