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Blood, 22 January 2009, Vol. 113, No. 4, pp. 866-874. Prepublished online as a Blood First Edition Paper on October 16, 2008; DOI 10.1182/blood-2007-12-124818.
MYELOID NEOPLASIA Very late antigen-4 function of myeloblasts correlates with improved overall survival for patients with acute myeloid leukemia1 Division of Hematology, University of Washington, Seattle; 2 Southwest Oncology Group Statistical Center, Fred Hutchinson Cancer Research Center, Seattle, WA; 3 Cancer Research and Treatment Center, University of New Mexico, Albuquerque; 4 Division of Medical Oncology, University of Washington, Seattle; and 5 Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA
Adhesion of acute myeloid leukemia (AML) blasts in the bone marrow microenvironment confers protection from chemotherapy-induced apoptosis. One mechanism for retention of blasts within the bone marrow is adhesion via very late antigen-4 (VLA-4), the  4β1 integrin heterodimer that binds to its main ligands, fibronectin, and vascular cell adhesion molecule-1 (VCAM-1). To examine the relationship of functional expression of VLA-4 to prognosis in AML, we studied marrow samples from 175 adult AML patients who underwent induction chemotherapy with anthracycline and cytarabine on Southwest Oncology Group trials. The studies included flow cytometry and functional in vitro assays for ligand binding and maximal β1 activation. VLA-4 expression varied widely, with mean expression 60.6% for 4, and was not significantly associated with response to chemotherapy, relapse-free, or overall survival (OS). However, increased binding of soluble VCAM-1 via VLA-4 was significantly associated with longer OS, corrected for age (P = .033). Estimated 5-year OS was 31% (95% confidence interval, 14%-48%) in 30 patients with soluble VCAM-1 binding greater than or equal to 40%, compared with 10% (confidence interval, 3%-17%) in 72 patients with lower binding. Adhesion and migratory properties of AML blasts thus appear to influence chemosensitivity and therefore may be therapeutic targets.
The majority of adult patients with de novo acute myeloid leukemia (AML) will achieve an initial complete remission, but with chemotherapy alone, most will relapse. Moreover, there are poor prognostic groups, such as treatment-associated leukemia or transformed leukemia, or those with poor prognosis cytogenetics, who are less probable to achieve complete remission with induction treatment and for whom the overall survival (OS) is less than a year.1 Normal stem/progenitor cell retention in favorable anatomic location or niches in the bone marrow is critical for their survival and further development. The receptor CXCR-4 is thought to play a critical role in retention of human cells to the bone marrow after transplantation through interaction with its ligand, stromal-derived factor-1 (SDF-1),2 which is highly expressed in niches within the bone marrow. In addition to CXCR-4/SDF-1, this retention is facilitated by several other cooperative pathways, such as that of very late antigen-4 (VLA-4) integrin and its major ligands within the bone marrow, vascular cell adhesion molecule-1 (VCAM-1), and fibronectin. Adhesive properties of leukemic cells are probably responsible for the complication of leukostasis in AML as well as leukemic meningitis, leukemia cutis, extramedullary leukemia, and formation of chloromas. Several adhesion mechanisms, including the VLA-4/VCAM-1 pathway, have been implicated in the attachment of leukemic blasts to the vessel wall.3 High levels of expression of VLA-4 are seen in all French-American-British classes of AML, M0-M5, with averages ranging from 72% to 95%, although there is a wide range (6%-98%).3 VLA-4 is involved in the migration of CD34+ cells and AML cells beneath marrow stromal cells.4 In addition, blocking of CXCR-4 by AMD 3100 results in mobilization of AML blasts into the circulation.5 Moreover, administration of an activating antibody to CD44, the hyaluronic acid receptor, which induces differentiation in vitro, blocks engraftment of AML cells in NOD-scid mice.6 Furthermore, administration of this antibody to CD44 can selectively eliminate the engrafted leukemia cells but has no effect on engrafted normal hematopoietic cells derived from cord blood or human bone marrow.6 Thus, multiple adhesion mechanisms, many of which are shared by normal cells, are probably critical for the movement to and retention of acute leukemia cells within a specific bone marrow microenvironment. Adhesion of cells has been demonstrated to confer resistance to several chemotherapy agents, including cytarabine and etoposide. Growth of AML cells on HS-5 stroma reduces daunorubicin-or cytarabine-induced apoptosis.7 Adhesion of U937 to fibronectin via β1 integrins inhibits mitoxantrone- and etoposide-induced apoptosis8; similarly, adhesion of U937 or HL60 leukemia cell lines to fibronectin inhibits daunorubicin- or cytarabine-induced apoptosis.9
Several potential mechanisms have been proposed for the ability of integrin-mediated signaling to protect from chemotherapy toxicity, including activation of the phosphatidylinositol 3-kinase (PI3K)/Akt/bcl-2 pathway9 and an interaction between Wnt and adhesion-dependent signaling pathways.10 Furthermore, a function blocking antibody to VLA-4 restored chemotherapy sensitivity to cytarabine in a murine xenograft leukemia model of minimal residual disease.9 These investigators also reported that 5-year survival was 100% for patients whose leukemia cells exhibited low expression of VLA-4 (< 34.5%), compared with 44% for patients whose leukemia cells exhibited high expression of VLA-4 ( To further explore the role of VLA-4 in survival in AML, we examined the expression and function of VLA-4 by flow cytometry, functional assays, and real-time quantitative reverse-transcribed polymerase chain reaction (RT-PCR) in 175 bone marrow samples obtained from patients with previously untreated AML, who subsequently received treatment on Southwest Oncology Group (SWOG) protocols with an anthracycline and cytarabine. The results of expression and function of VLA-4 were then correlated with response to induction chemotherapy, relapse-free survival (RFS), and OS.
Human subjects This study was conducted with approval of the Fred Hutchinson Cancer Research Center Institutional Review Board. Informed consent was obtained in accordance with the Declaration of Helsinki. Sample selection
We selected 175 cryopreserved pretreatment marrow specimens from patients with previously untreated AML who were eligible for and received anthracycline- and cytarabine-containing induction chemotherapy regimens on the following SWOG clinical trials: SWOG-8600, SWOG-9031, SWOG-9126, S9333, and S9500. Treatment regimens and clinical results of these trials have been reported separately.11–15 Eligible specimens had at least 3 vials of cryopreserved cells stored in the SWOG repository and probably had more than or equal to 15% blasts in the sample based on the repository's count, if available, or on the patient's clinical trial pretreatment data. All selected specimens had at least 27 million cells/vial, but this was not set as a minimal requirement. The selection included all eligible specimens from patients known to have prior myelodysplastic syndrome or treatment-related secondary AML. The remaining patients were randomly selected with stratification to ensure approximately equal numbers older and younger than age 56. The sample size of 175 was designed to ensure that a 2-sided test at the
Thawing of bone marrow samples Cryopreserved bone marrow samples received from the SWOG repository were kept frozen in liquid nitrogen until use. Samples were rapidly thawed by addition to prewarmed thaw media (2.5% bovine serum albumin [BSA] and 5% Dextran 40 from Sigma-Aldrich, St Louis, MO, with 50 U/mL DNAse I [Boehringer Ingleheim, Ridgefield, CT] in phosphate-buffered saline, sterile filtered). The cells were incubated in Iscove modified Dulbecco medium (IMDM), 15% heat-inactivated fetal bovine serum, 15% horse serum, 1% Pen-strep (IMDM, fetal bovine serum, and penicillin-streptomycin [Pen-strep] from Invitrogen, Carlsbad, CA); 49.5 µM β-mercaptoethanol and 49.4 µM monothioglycerol (Sigma-Aldrich); and interleukin-3 (IL-3; 100 ng/mL) and stem cell factor (SCF, 10 ng/mL) from R&D Systems (Minneapolis, MN) for 16 hours. Mononuclear cells were then isolated by density depletion and viable cells enumerated by trypan blue exclusion. Effect of freeze, thaw, and overnight incubation on VLA-4 expression The leukemia cell lines, HL60, MEG-01, and K562 (ATCC, Manassas, VA), and 2 patient AML samples were used to compare the effect of freeze/thaw and 16-hour incubation with low-dose SCF (10 ng/mL), SCF with IL-3 (100 ng/mL each), and thrombopoietin with Flt3/flk2 ligand (100 ng/mL each; cytokines from R&D Systems). VLA-4 expression was analyzed by flow cytometry. Flow cytometric analysis
Flow cytometric analysis for VLA-4 expression by the leukemia blast population was performed on each SWOG sample. After the samples were thawed, they were labeled with 1:50 dilutions of the following antibodies (0.2-0.5 million cells/tube): phycoerythrin-conjugated antibodies CD49d ( Functional adhesion assay to fibronectin For 20 patient samples, CD34+ blast cells were isolated using the VarioMACS (Miltenyi Biotec, Auburn, CA). Lab Tek 8-well chamber slides were coated with 8 µg/mL per cm2 recombinant fibronectin fragment, CH-296 (Retronectin; Takara, Kyoto, Japan). Nonspecific adhesion was assessed in control wells coated with 2% heat-inactivated BSA; 105 patient CD34+ AML cells were incubated in serum-free media for 2 hours. The wells were washed, fixed with 1% paraformaldehyde, and counterstained with 4',6 diamidino-2-phenylindole in Vectashield (Vector Laboratories, Burlingame, CA) and sealed. Ten fields per well were examined by fluorescence microscopy, and bound cells were enumerated. Binding of fluorescently labeled soluble VCAM-1 After study of the first 73 patient samples for VLA-4 expression, we introduced a flow cytometry assay to examine functional binding of soluble VCAM-1 (sVCAM-1) for the remaining 102 patients. sVCAM-1 (R&D Systems) was fluorescently labeled with 5-(and 6)-carboxyfluorescein ester (Invitrogen) and the concentration determined by A280 (for 1 mg/mL, A280 = 0.84). A total of 2 x 105 AML cells/patient sample were incubated with 1 µM fluorescently labeled sVCAM-1, washed, and fixed. Nonspecific binding was determined as described.18 Percentage binding and MFI for specific and nonspecific binding were obtained. Expression of activation-dependent epitope of β1 The expression of functionally activated β1 in 24 selected patient samples was assessed with 9EG7 antibody (BD PharMingen) before and after treatment with activating antibody 8A2.19 Then cells were labeled with 9EG7 antibody recognizing the activated epitope. Cells were then analyzed by flow cytometry. Real-time RT-PCR for VLA-4
CD34+ blasts from 23 randomly selected patient samples were isolated using the VarioMACS. RNA was isolated using the RNeasy Kit and the RNAse-Free DNase set (QIAGEN, Valencia, CA). Reverse transcription was performed using the Reverse Transcriptase iSCRIPT cDNA Synthesis Kit (Bio-Rad, Hercules, CA) and real-time polymerase chain reaction (PCR) performed using Chemotherapy resistance assays
We examined the ability of fresh and previously frozen primary AML cells to exhibit chemotherapy resistance afforded by plating on Retronectin or immobilized VCAM-1. These patient cell samples were obtained on the same Institutional Review Board–approved protocol but were not from the SWOG repository. Cells were preincubated with no azide, low endotoxin Statistical analysis The following clinical information was available for analysis: WBC count at diagnosis, age, French-American-British classification, flow cytometry phenotype, cytogenetics, response to induction therapy (CR and resistant disease), time to CR, RFS (measured from CR until relapse or death from any cause, censored at last contact for patients last known to be alive without report of relapse), and OS (measured from study entry until death from any cause, censored at last contact for patients last known to be alive). Karyotypes were classified as favorable, intermediate, or unfavorable according to previously published criteria (Table S1Table S1).20 Data regarding FLT3–internal tandem duplication (ITD) (present vs absent, and ITD length) were available for 77 patients from a previous study (Table S2Table S2).21 We examined VLA-4 expression by flow cytometry for all 175 patients, and for subgroups examined functional adhesion to fibronectin, sVCAM-1 binding, and expression by quantitative real-time RT-PCR. Nonparametric methods were used to compare expression levels and MFIs between groups (Wilcoxon rank sum test) and to estimate correlation coefficients (Spearman R). Distributions of RFS and OS were estimated by the method of Kaplan and Meier.22 Multivariate analyses, based on logistic regression models (CR, resistant disease) or proportional hazards regression models (OS, RFS), were performed to examine the prognostic significance of VLA-4 and sVCAM-1 expression and function in the presence of other prognostic factors. Statistical significance was characterized by 2-sided P values. Analyses were based on data available April 5, 2007.
Effect of thawing and cytokines on VLA-4 expression
No significant differences were observed in the percentage expression of VLA-4 flow cytometric analysis
VLA-4 expression varied widely among the 175 patients, with a mean expression of 60.3% (range, 5.4%-99.7%) for
Function of VLA-4: in vitro adhesion to recombinant fibronectin peptide and sVCAM-1 binding
Because VLA-4 expression does not ensure function, we wished to directly assay for functional VLA-4. We began with a standard in vitro adhesion assay, but this required isolation of the blast population, which could only be performed for the patients who exhibited high expression of CD34, with sufficient cell numbers for both the separation and subsequent assay. This in vitro adhesion assay assessed binding of VLA-4 to its 2 ligands, fibronectin and VCAM-1, coated on tissue-culture wells, for a subsample of 20 patients. Flow cytometric measures of
We then switched to the flow cytometry method for sVCAM-1 binding, which could be performed for all patient samples, because the function could be assessed on the gated blast population. Using this second functional assay for 102 patient bone marrow samples, the mean percentage binding and MFI of sVCAM-1 were 28.6% (range, 2.0%-96.8%) and 83.1 (range, 10.4-520.1), respectively. The percentage binding of sVCAM-1 increased with increasing Activated β1 analysis
β1 integrin, which combines with
Quantitative real-time PCR for VLA-4
Because there have been several recent studies of RNA expression profiling in AML, we sought to determine whether there was a correlation between level of mRNA for Correlation of VLA-4 expression with patient characteristics and treatment outcomes
Maximal activated β1 expression, as determined by staining with 9EG7 antibody after pretreatment with 8A2, decreased sharply with increasing age (R = –0.83, P < .001) (Figure 3), whereas unstimulated β1 expression as a fraction of this maximum increased somewhat with age (R = 0.33, P = .12).
Among the 102 patients with sVCAM-1–binding data, sVCAM-1 MFI reflecting density of binding sites decreased with increasing age (R = –0.30, P = .002), although sVCAM-1 percentage binding did not (R = –0.11, P = .29). Among the 59 patients with sVCAM-1 binding data and known AML onset (primary or secondary), sVCAM-1 percentage binding, MFI, and combined expression were all somewhat lower in those with secondary AML (Wilcoxon P = .024, .13, and .045, respectively). Eighty-four (48%) of the 175 patients achieved CR. There were no significant associations between CR rate and any of the measures of VLA-4 expression or function. The CR rate was somewhat higher for patients in the upper quartile of sVCAM-1 binding, compared with those with lower levels of sVCAM-1 binding (64% vs 44%), although the trend was not statistically significant (P = .18, adjusted for the effect of age on CR rate). Of the 175 patients, 148 have died and the remaining 27 were last known to be alive after 5 months to 13.8 years (median, 9.2 years). There were no clearly significant associations between OS and any of the measures of VLA-4 expression or function (Figure 4). However, OS increased with increasing percentage binding of sVCAM-1 (P = .023 for percentage expression, Figure 5). In multivariate analyses adjusting for the effect of age on OS, the effect of sVCAM-1 binding persisted (P = .033). This effect was largely the result of patients whose sVCAM-1 binding was in the highest quartile for whom the estimated mortality hazard ratio, relative to the lowest quartile of sVCAM-1 binding, was 0.57 (confidence interval, 0.30-1.07). The effect of sVCAM-1 binding on OS also persisted in multivariate analyses that adjusted for the effects of cytogenetic risk classification or FLT3-ITD status (Table S3Table S3).
In vitro studies overcoming adhesion-mediated chemotherapy resistance by anti VLA-4 antibody or sVCAM-1 Primary AML cells from 3 patients pretreated with IgG isotype or left untreated showed increased viability in the presence of chemotherapy when plated on retronectin or immobilized VCAM-1, compared with cells plated on BSA control. Viability improved by an average of 28% on retronectin and 24% on VCAM-1, compared with BSA control (data not shown). In addition, apoptotic activity assayed by annexin staining decreased by an average of 29.5% on retronectin, and 22.5% on VCAM-1, compared with BSA control (data not shown).
However, the "protection" afforded by the VLA-4 interaction diminished when cells were pretreated with either
Both VLA-4 and CXCR-4 have been proposed as mechanisms for retention of AML blasts in the marrow. Functional inhibition of either of these receptors has been shown to dislodge AML cells and enhance chemotherapy-induced apoptosis in vitro and improve eradication of leukemia in vivo.9,25 To study the role of VLA-4 expression by AML blasts and its relevance in clinical outcome of AML, we analyzed VLA-4 expression and function in bone marrow samples from 175 adult AML patients who received anthracycline- and cytarabine-containing induction chemotherapy regimens. This study's use of banked cryopreserved cells must be considered when interpreting these results. Patients for whom samples were available were not representative of all patients on the clinical trials from which they were selected and, in particular, tended to have higher WBC and blast counts than excluded patients. This limits the generalizability of these results but not their validity. In addition, the use of cryopreserved samples necessitated thawing of the cells before examination. AML cells undergo apoptosis in culture without media containing serum and cytokines24; therefore, AML cell viability was maintained by incubating cells in media supplemented with serum, SCF, and IL-3. With the short overnight incubation time and dose of cytokines used, we did not observe changes in VLA-4 expression, as demonstrated by comparing various cytokine concentrations and fresh versus frozen AML samples. This is consistent with our previous findings showing that a longer incubation time in cytokines was required to affect VLA-4 expression in HL60 cells.26
By means of a successive gating strategy, the flow cytometry analyses were restricted to viable AML blasts (Figure S1Figure S1). β1 (the main partner of
For all integrins, expression does not imply function, and only activated integrins have binding capacity. To test the functional status of
A significant correlation between binding of sVCAM-1, reflecting functional activity of VLA-4, and OS in AML was documented, with the mortality rate reduced by 43% in the highest quartile of sVCAM-1–binding MFI, compared with the lowest quartile. In contrast to a previous report,9 we found no clearly significant associations of VLA-4 percentage expression with response to chemotherapy or with disease-free survival or OS (Figure 4). Notably, Matsunaga et al reported no deaths in 10 patients they classified as negative for Our studies clearly demonstrated that sVCAM-1 was as effective as anti–VLA-4 antibody in restoring sensitivity to chemotherapy in vitro for leukemia cells attached to immobilized VCAM-1 or retronectin. This observation allows the formulation of a putative scenario to explain the pronounced effect of sVCAM binding on survival. If, within the bone marrow microenvironment, sVCAM-1 is increased (released by leukemic cells or by chemotherapy induced cell lysis), the VLA-4–expressing leukemic cells bound to sVCAM-1 will be dislodged, enhancing the effect of chemotherapy. This movement would be similar to the migration of lymphocytes induced by sVCAM-1,29 although the leukemia cells would not necessarily need to leave the bone marrow to undergo apoptosis. Others have reported that plasma levels of sVCAM-1 are elevated in AML patients compared with healthy controls (P < .001).30 In that report, increased levels of either sE-selectin or sVCAM-1 were present in 32 of 40 leukemic patients (80%) and the highest levels were measured in M4-AML.29 We did not measure plasma sVCAM-1 levels in our study. The exact mechanism by which blocking of adhesion leads to increased chemosensitivity is uncertain. Matsunaga et al demonstrated that binding of AML blasts to fibronectin via VLA-4 resulted in signaling through the PI3K/Akt pathway, leading to increased bcl2 and enhanced survival when the leukemia cells were adherent.9 Antibody blocking of VLA-4, PI3K inhibitors, or antisense oligonucleotides to bcl-2 all restored chemotherapy-induced apoptosis. It is also possible that there may be alternative downstream signaling pathways for VLA-4, analogous to the distinct pathways induced by the Flt3 tyrosine kinase domain mutations as opposed to the Flt3-ITDs.31 Moreover, there are countless other potentially contributing influences; for example, Flt3 ligand itself has been shown to enhance adhesion via VLA-4,32 and Flt3-ITDs may enhance the expression of CXCR-4, involved in marrow homing/retention through binding of SDF-1, and thought to convey poor prognosis in AML.33–35 These data suggest that sVCAM-1 binding by AML blasts may have value as a prognostic parameter for OS at diagnosis. It is evident that it is not simply adhesion receptor expression, but the functional interactions with stromal cells and other cells in the bone marrow microenvironment that may ultimately determine the susceptibility to chemotherapy, protection from apoptosis, or migratory behavior of leukemic cells. Disruption of the adhesion pathways on which leukemia cell survival is dependent might be of therapeutic benefit, and clinical investigations in this direction are underway. Prospective study of functional VLA-4 expression and response to treatment is warranted.
Contribution: P.S.B. designed and performed research, analyzed data, and wrote the paper; K.J.K. designed research, analyzed data, and wrote the paper; A.N.W. performed research, analyzed data, and wrote the paper; S.C. performed research, contributed to study design, and analyzed data; J.M.H. contributed vital reagent and contributed to study design; C.L.W. contributed repository samples and methodology and contributed to study design; S.H.P. contributed to study design and patients for study; D.L.S. contributed data; and T.P. and F.R.A. designed research and wrote the paper. Conflict-of-interest disclosure: The authors declare no competing financial interests. Correspondence: Pamela S. Becker, University of Washington, Box 358056, 815 Mercer Street, Seattle, WA 98109; e-mail: pbecker{at}u.washington.edu.
We thankfully acknowledge the Southwest Oncology Group Leukemia Repository for providing the patient bone marrow samples. We are grateful to Dr Erica Jonlin for assistance with the regulatory documents. We acknowledge Dr Xin Zhao for technical assistance and data analysis. This work was supported by Southwest Oncology Group Development Funds (National Cancer Institute), a grant from the Leukemia and Lymphoma Society Translational Research Program (P.S.B.), and the Douglas Kroll Research Foundation.
Submitted December 10, 2007; accepted September 24, 2008.
Prepublished online as Blood First Edition Paper, October 16, 2008
DOI: 10.1182/blood-2007-12-124818
The online version of this article contains a data supplement.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 USC section 1734.
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Abstract
Introduction
34.5%),
) or Ara-C plus daunorubicin (N = 2, 
) was an average of 26 plus or minus 7 (SE) percentage points lower for cells preincubated with 
18.6%), who had median follow-up more than 3 years,
