|Year : 2013 | Volume
| Issue : 3 | Page : 108-114
Expression and significance of osteopontin and its receptor CD44v6 in acute myeloid leukemia
Nihal M. Heiba1, Shereen A. Elshazly2
1 Department of Clinical Pathology, Faculty of Medicine, Ain Shams University, Cairo, Egypt
2 Department of Internal Medicine, Faculty of Medicine, Ain Shams University, Cairo, Egypt
|Date of Submission||14-Mar-2013|
|Date of Acceptance||04-Apr-2013|
|Date of Web Publication||19-Jun-2014|
Nihal M. Heiba
MD, Departments of Clinical Pathology, Faculty of Medicine, Ain Shams University, Ramses St, Abbassia 11566, Cairo
Source of Support: None, Conflict of Interest: None
The bone marrow niches are proposed to be the supportive microenvironment that offers cytoprotection from chemotherapy and confers a survival advantage to leukemic cells that could be responsible for relapses in patients with acute myeloid leukemia (AML). Osteopontin (OPN) and its receptor CD44v6 play a crucial role in leukemia cell homing to the bone marrow.
This study aimed at investigating the pattern of expression and prognostic impact of OPN and CD44v6 in AML patients and their correlation with each other.
Patients and methods
The study was carried out on 70 patients with de-novo AML and 20 age-matched and sex-matched controls. Patients were diagnosed and classified according to the French American British classification/WHO (FAB/WHO) criteria by cytomorphology, immunophenotyping, and cytogenetic analysis. Cellular OPN and surface CD44v6 expression by blast cells was assessed by flow cytometry.
OPN was overexpressed by AML blasts (8.2–75.8%; median 25%) compared with the controls (<5%), with 32/70 (45.7%) showing higher levels than the median (>25%). CD44v6 was detected in 56/70 (80%) patients, with 30/70 (42.9%) showing high expression (≥20%). OPN expression was correlated positively with that of CD44v6, its overexpression being paralleled by an increase in that of CD44v6. Higher levels of OPN and CD44v6 were not related to patients’ clinical or laboratory data, FAB subtype, cytogenetic risk group, and initial response to primary induction chemotherapy. A shorter event-free survival was observed in patients with higher OPN and CD44v6 expression (4 months) compared with those with lower levels (16 months), and elevated OPN and CD44v6 at diagnosis were each identified as a negative independent prognostic marker.
OPN and its receptor CD44v6 levels of expression are increased in AML, correlate with each other, and their higher levels at diagnosis can be used as early prognostic markers to predict poor disease progression.
Keywords: acute myeloid leukemia, CD44v6, osteopontin
|How to cite this article:|
Heiba NM, Elshazly SA. Expression and significance of osteopontin and its receptor CD44v6 in acute myeloid leukemia. Egypt J Haematol 2013;38:108-14
|How to cite this URL:|
Heiba NM, Elshazly SA. Expression and significance of osteopontin and its receptor CD44v6 in acute myeloid leukemia. Egypt J Haematol [serial online] 2013 [cited 2020 Jan 23];38:108-14. Available from: http://www.ehj.eg.net/text.asp?2013/38/3/108/134787
| Introduction|| |
Acute myeloid leukemia (AML) has long been considered as a malignant hierarchical disease with cell-autonomous mutations that are causally implicated in disease pathogenesis. Nowadays, there is compelling emerging evidence that in AML, as well as other hematologic malignancies, not only molecular events inside the malignant cell clone but also their interactions with the microenvironments play a pivotal role in disease maintenance and progression 1. Osteopontin (OPN) and CD44v6 are among the molecules implicated in these interactions.
OPN is a secreted glycoprotein of the SIBLING-family that is encoded by a single gene on chromosome 4q13 and through phosphorylation, glycosylation, and proteolytic cleavage, various molecular masses, from 25 to 75 kDa, arise. It is synthesized by many cell types and is involved in many physiological and pathological processes, including cell adhesion, angiogenesis, apoptosis, inflammatory responses, and tumor metastasis, with the ability to serve a dual function as a chemoattractant cytokine and as an extracellular component 2,3. In the normal bone marrow (BM), OPN is most prominently expressed in the osteoblasts lining the bone trabeculae and acts as a key regulator of bone hemostasis 4. It also plays an important role in hematopoietic stem cell (HSC) regulation and hematopoiesis by contributing to HSC-transmarrow migration toward the endosteal region and maintaining their quiescence by inhibiting entry into the cell cycle 2. As OPN has been strongly associated with tumorogenesis and worsened survival in solid tumors 5, recently, a few reports have implicated it in hematological malignancies such as multiple myeloma 6,7 and chronic myeloid leukemia 8,9.
The wide range of effects elicited by OPN is attributable to its various forms and its multiple receptors and binding sites, such as multiple integrins (αvβ3, α9β1, αvβ7) 3 and CD44 through the v6 and v7 variants 10.
CD44v6 is one of many isoforms of CD44 generated by alternative splicing of the gene on the short arm of chromosome 11. CD44 is a transmembrane glycoprotein with an extracellular domain that binds many ligands as chondroitin acids. It is expressed ubiquitously and involved in lymphocyte homing and activation and adherence of various cells including fibroblasts and CD34+ hematopoietic progenitors to extracellular matrix. CD44v is far more restricted in normal tissues and the v6 isoform has been detected on monocytes, macrophages, and activated T cells 11. CD44v6 has been reported to be upregulated in non-Hodgkin lymphoma 12 and multiple myeloma 13, correlating with an unfavorable clinical evolution.
In the present study, we aimed to investigate the expression of OPN and its receptor CD44v6, together with their clinical and prognostic implications and their correlation with each other, in AML patients.
| Patients and methods|| |
This study was carried out on leukemic cells of 70 newly diagnosed AML patients from the Hematology/Oncology Unit, Ain Shams University Hospital, in the period between February 2011 and July 2012. There were 41 men and 29 women (M : F=1.4 : 1) ranging in age from 18 to 65 years (mean±SD=35.7±15.4). Their main clinical features are summarized in [Table 1]. BM sampled obtained from 20 age-matched (34.1±12.3) and sex-matched (M : F=1.3 : 1) individuals with nonhematological malignancies (e.g. patients with hypersplenism or immune thrombocytopenia) served as controls.
The study was approved by the local Research Ethics Committee and a written informed consent was signed by all participants before enrollment.
Patients were subjected to assessment of full history, clinical examination, and radiological investigations. Diagnosis was confirmed using peripheral blood (PB) and BM aspiration biopsy samples for the following: (i) complete blood count (Coulter LH750; Beckman Coulter Inc., Fullerton, California, USA); (ii) cytomorphology of Leishman and immunohistochemically stained PB and BM smears; (iii) leukocyte antigen expression using a panel of phycoerythrin (PE)/fluorescein isothiocyanate-conjugated monoclonal antibodies (moAb) to HLADR, CD34, CD13, CD14, CD15, CD33, cytoMPO, CD61, CD117, glycophorin A, CD2, CD3, CD5, CD7, cytoCD3, CD10, CD19, CD20, and CD79a with their isotype-matched controls (Coulter Electronics, Hialeah, Florida, USA); and (iv) cytogenetics using the conventional G-banding technique according to standard protocols 14 and FISH analysis in line with the procedures described by Vance et al. 15 using probes for t(8;21), t(15;17), t(9;22), inv(16), −5, and −7 (Vysis, Downers Grove, Illinois, USA).
Classification was performed according to the FAB and WHO revised criteria of myeloid neoplasms 16 and cytogenetic risk categorization was in line with the Medical Research Council schema 17. Patients with t(8;21), t(15;17), or inv(16) were considered to have favorable risk cytogenetics; those with −5, del(5q), −7, abnormalities of 3q or complex cytogenetics were classified into the unfavorable-risk group; the intermediate-risk group included patients with normal cytogenetics or other miscellaneous single abnormalities.
Patients were treated with standard induction chemotherapy (combination of Adriamycin 45 mg/m2 on days 1–3 and cytosine arabinoside 100 mg/m2/day on days 3–9), followed by three cycles of consolidation with high-dose cytarabine after achievement of complete remission (CR) 18. Unfavorable-risk cytogenetic patients were scheduled for HSC transplantation after CR. PML/RARα-positive M3 patients were treated according to the PETHEMA protocol (induction by all-trans retinoic acid 45 mg/m2/day until CR with idarubicin 12 mg/m2 on days 2, 4, 6, and 8 and consolidation according to protocol) 19.
Patients were followed up over 18 months (median 9 months); complete blood count and BM aspiration examination were performed to assess response to treatment according to the revised National Working Group criteria proposed by Cheson et al. 20.
Morphologic CR criteria were as follows: (i) PB neutrophil count more than 1.0×109/l, platelet count more than 100×109/l, and independence of red cell transfusion; (ii) absence of blasts in PB; (iii) less than 5% blasts, not in clusters or collections, and no detectable Auer rods in a BM sample with normal maturation of all cell lines; and (iv) no extramedullary leukemia. Incomplete response was defined as 5–25% blasts in BM with a decrease in BM pretreatment blast percentage by at least 50% or less than 5% blasts in the presence of Auer rods in a BM with normal maturation and a PB without leukemic cells. Resistance referred to no response on the basis of the presence of blasts and absence of hypocellularity.
For the determination of OPN and CD44v6 expression by flow cytometry (FCM), fresh BM or PB samples were obtained on EDTA-anticoagulated vacutainer tubes and were processed within 6 h. In cases of unavoidable delay, samples were preserved at room temperature (22–24°C) for a maximum of 24 h. BM samples (1 : 3) and PB samples (1 : 1) were diluted with PBS, pH 7.4, and the final cell count was adjusted to 5–10×109/ml to be processed according to the whole-blood lysis method.
Intracytoplasmic detection of osteopontin by flow cytometry
Cellular staining of OPN is limited to the cytoplasm; therefore, cells were first fixed and permealized in two steps: first in 3.5% paraformaldehyde/PBS and then in 50% cold acetone/PBS. This was followed by the addition of 10 μl of antihuman OPN, PE-conjugated moAb (R&D Systems Inc., UK), to be incubated for 30 min in the dark at room temperature. Cells were washed in PBS and red cell lysis was carried out using a 1.5 ml NH4Cl solution buffered with KHCO3 at pH 7.2 for 3 min at room temperature in the dark, followed by wash in PBS. Analysis was carried out on a Coulter Epics XL Flow Cytometer (Coulter Electronics). In cases of normal control BM samples, various populations of cells (lymphocytes, monocytes, and granulocytes) were gated on the basis of labeling by phycoerythrin cyanine 5-labeled anti-CD45 (Beckman Coulter Inc.), whereas blast cells in cases of AML were gated on a forward scatter/side scatter dot-plot histogram, followed by determination of the percentage of cells expressing OPN relative to negative isotype-matched antibody controls.
Detection of surface CD44v6 expression by flow cytometry
Staining for CD44v6 is expressed on the surface of cells, but initial Fc-blocking of cells is essential. Cells were Fc-blocked by previous treatment with 1 μg human IgG/105 cells for 15 min at room temperature. No wash was performed and 50 μl of whole blood was mixed with 10 μl of PE-conjugated antihuman CD44v6 moAb (R&D Systems Inc.) and incubated for 30–45 min at 2–8°C. Cells were washed with PBS, followed by red cell lysis as described. Data of cells in normal BM controls and blasts of AML samples were acquired on the flow cytometer in the same manner described for OPN and the percent of cells positive for CD44v6 was determined relative to the negative isotype-matched control. A sample was considered negative (−) when less than 5% of the cells were labeled, positive (+) for 5 to less than 20% of labeled cells, and highly positive (++) when 20% or more of the cells expressed CD44v6 21.
Continuous variables were reported as median or mean±SD and comparison between groups was carried out using the Mann–Whitney U-test (for nonparametric data) and Student’s t-test for parametric data. Categorical variables were expressed as number and % and differences among groups were assessed using Fischer’s exact test. Correlation between two variables was determined using the Spearman rank correlation coefficient. Event-free survival (EFS) rates for patients receiving conventional chemotherapy, from the start of treatment to relapse or death, were estimated using the Kaplan–Meier method and compared using the log-rank test. Patients alive in CR at the time of last contact or at the end of the study period were censored, and a relapse or death was marked as an event. Multivariate analysis using the Cox proportionate hazard regression model was used to identify significant prognostic factors on the basis of EFS. A P-value of less than 0.05 was considered statistically significant and P-value less than 0.01 as highly significant, all tests being two-tailed. Calculations were carried out using Graphpad Prism Software, version 4.0 (Graphpad Software Inc., La Jolla, California, USA).
| Results|| |
In all normal control BM samples, OPN was expressed by less than 5% of all the cells tested.
In AML patients, OPN was expressed by blast cells ranging from 8.2 to 75.8% (median 25%), being significantly higher than that in the control samples (P<0.01).
On considering the median value (25%) as a cut-off level to categorize patients into low OPN (⩽25%) or high OPN (>25%) expressors, 32/70 (45.7%) had higher OPN expression levels. These higher OPN levels did not significantly relate to any of the patients’ clinical information (age, sex, organomegaly), laboratory data (Hb level, total leukocyte count, platelet count, PB, or BM blasts), or FAB subtype and cytogenetic risk group [Table 2].
|Table 2: Relation of osteopontin and CD44v6 expression levels to known prognostic indicators in acute myeloid leukemia patients|
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Control BM samples were all negative for CD44v6 (<5% expression).
CD44v6-expressing leukemic cells were detected in 56/70 patients (80%), ranging between 5.9 and 80.3%, 26 of them being weakly positive (<20%) and 30 highly positive (≥20%). M4–5 patients had the highest median level of expression (38.1%), M0–1 had 18.6%, M2 had 22.9%, and M3 had 28.4%, although not reaching statistical significance (P>0.05).
Patients were categorized according to CD44v6 expression: 40/70 (57.1%) had negative/low positive (−/low+) CD44v6 and 30/70 (42.9%) had highly positive (++) CD44v6. High levels of CD44v6 did not relate to the clinical or the laboratory data of the patients, FAB subtype, and cytogenetic risk group [Table 2].
Correlation of osteopontin and CD44v6 expression
The cellular expression of OPN was highly significantly positively correlated with CD44v6 surface expression of leukemic cells in AML patients (r=0.88; P<0.01) [Figure 1]. Twenty-five of 32 (78%) patients with higher OPN expression had highly positive CD44v6 levels and 33/38 (86%) with lower OPN levels were −/low+ for CD44v6, whereas only 12/70 (17%) patients expressed OPN and CD44v6 disconcordantly.
|Figure 1: Correlation of osteopontin (OPN) and CD44v6 expression in acute myeloid leukemia. CD44v6 expression is correlated positively with that of OPN (r=0.88; P<0.01).|
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Relation of osteopontin and CD44v6 expression to patient outcome
Response to primary induction chemotherapy
Ten of the 70 AML patients included in the study died early during the course of the disease either before the start or at the beginning of the induction treatment. Fifty (71%) achieved CR after the first induction chemotherapy course and 10 (14.3%) were either resistant or partial responders. Neither of the OPN or CD44v6 higher levels of expression exerted an effect on the response to therapy at day 28 [Table 3].
|Table 3: Relation of osteopontin and CD44v6 expression to response to initial induction chemotherapy and overall conventional chemotherapy outcome in acute myeloid leukemia patients|
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Overall response to treatment
Ten of the patients who achieved CR were excluded from the study as they had undergone HSC transplantation. Only 50 patients receiving conventional chemotherapy were further studied. Sixteen of 50 (32%) remained in CR throughout the observation period, 13 (26%) experienced a relapse, and 21 (42%) died. Twenty-two (44%) were high OPN expressors and 23 (46%) were high CD44v6 expressors.
On the basis of OPN levels of expression, patients with OPN more than 25% showed less tendency to remain in CR (3/22; 13.6%) compared with patients with lower OPN levels (13/28; 46.4%) (P<0.01) [Table 3]. The median EFS time (no relapse or death) for high OPN expressors was 4 months versus 16 months for patients with lower OPN expression levels (P<0.01). Kaplan–Meier and log-rank test showed that AML patients with lower OPN levels showed a better EFS probability than those with higher OPN levels (P<0.01) [Figure 2]a. Multivariate analysis showed higher levels of OPN at diagnosis to be a negative prognostic factor, independent of other established risk parameters, in AML (HR=−3.4; P<0.01).
|Figure 2: Kaplan–Meier event-free survival (EFS) curves for (a) osteopontin (OPN) expression and (b) CD44v6 expression in acute myeloid leukemia patients. High OPN expressors have significantly shorter EFS compared with low OPN expressors (P<0.01). High CD44v6 expressors have significantly shorter EFS compared with CD44v6 −/low+ expressors (P<0.01).|
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Similarly, negative or low expression of CD44v6 in patients was linked to a significantly higher frequency to remain in CR (12/27; 44.4%) compared with highly positive CD44v6 patients, of whom only 4/23 (17.4%) remained in CR throughout the follow-up period, and 19/23 (82.6%) either experienced a relapse or died (P<0.01)[Table 3]. Kaplan–Meier analysis and log-rank test for EFS showed that patients with higher expression of CD44v6 had a higher tendency to experience relapse or die, their median EFS time (4 months) being significantly lower than that for patients with −/low+ CD44v6 (16 months; P<0.01) [Figure 2]b]. In the Cox-regression analysis, higher levels of CD44v6 expression at diagnosis emerged as a negative independent prognostic risk factor (HR=−3.2; P<0.01).
| Discussion|| |
AML is highly sensitive to chemotherapy, but after initial responses, relapses often occur and the long-term survival of patients is dismally poor, being 30–40% at 4 years after diagnosis. Leukemia relapse is believed to be sustained by a subset of leukemia cells that are chemoresistant and reinitiate the disease, and the endosteal region of the BM (or the BM niche) was identified as the site of homing and protective microenvironment for the chemotherapy-resistant AML cells 22. There is growing evidence that a complex interplay of signals within the BM microenvironment imprints a ‘chemoresistant phenotype’ to AML cells; among these, OPN and its receptor CD44v6 are believed to play crucial roles in leukemia cell homing to BM 1.
In the present study, we investigated the expression of OPN and CD44v6 in AML blasts by FCM, which is a feasible, readily available technique, routinely applied to all AML patients at diagnosis.
Our data indicated OPN to be readily expressed by AML blasts of all patients included in the study compared with control participants. Higher levels of OPN expression (taking the median level as a cut-off) were found in 45.7% of the patients, not being related to patients’ clinical (age, sex, and organomegaly) or laboratory data (Hb level, total leukocyte count, platelet count, PB, or BM blasts), FAB subtype, and cytogenetic risk classification. These higher levels did not seem to affect the initial response of patients to primary induction chemotherapy. However, patients with higher OPN levels of expression at diagnosis were found to have a higher tendency toward a more aggressive disease course as evidenced by a shorter EFS (relapse or death; median 4 months) compared with low OPN expressors (median EFS 16 months); in addition, a higher initial OPN level was recognized as a negative independent prognostic factor by multivariate analysis. Similar findings have been reported by investigators applying different methods of OPN assessment in AML patients: Lee et al. 23, using ELISA for OPN in BM plasma, and Powel et al. 24, using RT-PCR, and Lierch et al. 25, using immunohistochemistry and mRNA level for microarray data of AML blasts.
The mechanism by which OPN seems to confer a more aggressive disease behavior on AML appears to be multifactorial and should be further investigated extensively. One possible explanation is that the physiologically negative regulatory role played by OPN in HSC proliferation under steady-state conditions could implicate that suppression of normal residual HSC may occur as a consequence of OPN overexpression in AML 3. In addition, OPN is documented to have a prosurvival and/or a proliferative function on various cells as well as an angiogenic role 26. OPN produced by tumor cells is considered to play a role in tumor growth and metastasis through enhancement of angiogenesis through an αvβ3-mediated, VEGF-enhanced pathway for endothelial cells (ECs) cytoprotection and induction of proliferation and motility 27,28. In agreement with this, Wierzbowsk et al. 29 observed that serum OPN levels in AML patients positively correlated with numbers of circulating ECs deriving from angiogenic microvessels.
The prosurvival activity of OPN was attributed to its binding to the CD44 variant isoform, leading to activation of the PI3K/AKT kinase cascade, which could be the reason why a large variety of malignant cells, including AML blasts, have evolved into producing increased levels of OPN as well as enhancing its production by other host cells (as stromal cells, osteoblasts, and osteoclasts) to acquire a growth advantage in an autocrine/paracrine manner 7, 9, 30.
Furthermore, it has been shown that OPN, by binding to the αvβ3 integrin receptor, enhances plasma membrane CD44v6 expression, with the resultant promotion of tumor metastatic behavior in hepatocellular carcinoma 31 and murine leukemia cells 32. To our knowledge, the present work is the first report on the correlation of OPN and CD44v6 expression in AML and it shows that OPN expression correlates positively with the surface reactivity for CD44v6 in AML blasts.
In this study, the CD44v6 expression, examined by FCM, was found to go almost hand in hand with that of OPN, being concordant in 83% of cases. CD44v6 was expressed by 56/70 (80%) of AML patients, being highly positive in 30 patients (42.9%), with a slight preference toward the M4–5 subtype, although not reaching statistical significance. This preference is expected as CD44v6 is normally present on monocytes 11,21. Higher levels of CD44v6 were not related to patients’ clinical or laboratory data and cytogenetic risk group. Despite the fact that high CD44v6 expressors did not differ in their initial response to primary induction chemotherapy from −/low+ CD44v6 expressors, higher levels of CD44v6 at diagnosis appear to adversely influence the later course of the disease, as evidenced by the shorter EFS periods (median 4 months) experienced by high expressors compared with −/low+ expressors (median 16 months). Moreover, multivariate analysis identified initial higher CD44v6 level as an independent unfavorable prognostic risk factor.
Legras et al. 21 reported findings comparable to ours, although CD44v6 was expressed by slightly fewer patients (71%), but with a similar impact on disease course. However, Bendall et al. 33 found CD44v6 to be less frequently expressed by AML blasts using FCM, but was present in 70% of cases when tested by PCR. This could be attributed to the fact that they examined a smaller number of patients (n=30) applying a higher threshold for positivity (10%) in FCM and that the mRNA may be present before the expression of the protein on the cell surface.
The relation of higher CD44v6 levels to a more aggressive disease course, evidenced by the shorter EFS and accelerated relapse, could be attributed to the role played by the CD44v6/ligand in AML cells migration, retention, and chemotherapy-protected survival in the BM niches 22. Moreover, the leukemic cells could possibly acquire a lymphocytic disguise, from overexpressed CD44v6 (as activated lymphocytes normally express CD44v6), and thus escape the recognition and killing by the immune system, allowing for more invasion and metastasis 11.
With the accumulating evidence of the increasingly important role played by the complex interactions between leukemic stem cells of AML and their niche microenvironment in the repopulating potential and ability to propagate and maintain the leukemic phenotype, it is worth pursuing novel treatment mechanisms targeting disruption of this adhesion 22. In fact, targeting of CD44 eradicates human myeloid leukemia stem cells in mice 34, and it is possible that targeting OPN may represent an important avenue for the development of therapeutics that block deregulated survival in AML 24 or block the proangiogenic effect on ECs more effectively than anti-VEGF 28.
In conclusion, the present study documents the increased expression of OPN and its receptor CD44v6 in AML blasts, with their higher levels of expression at diagnosis being able to define a subset of patients with an unfavorable disease course and shorter EFS. It also shows that OPN overexpression in AML is paralleled by an increased surface reactivity for CD44v6. Further larger scale studies are warranted to investigate this and to test the possibility of the use of OPN and CD44v6 levels at diagnosis as early biomarkers of disease aggression with their possible utilization as targets for ‘case-tailored’ AML therapy.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]