|Year : 2014 | Volume
| Issue : 3 | Page : 139-142
Circulating endothelial progenitor cells as a prognostic marker in childhood acute lymphoblastic leukemia
Hany A Labib MD, PhD 1, Ahmed G Siam2
1 Department of Clinical Pathology, Faculty of Medicine, Zagazig University, Zagazig, Egypt
2 Department of Pediatric, Faculty of Medicine, Zagazig University, Zagazig, Egypt
|Date of Submission||15-Oct-2014|
|Date of Acceptance||02-Nov-2014|
|Date of Web Publication||31-Dec-2014|
Hany A Labib
Clinical Pathology Department, Faculty of Medicine, Zagazig University, 9 Taleat Harb Street, Zagazig 44155
Source of Support: None, Conflict of Interest: None
Introduction Angiogenesis has been associated with the growth, the dissemination, and the metastasis of many tumors; it may also enhance the survival, the proliferation, and the chemoresistance of leukemic blasts cells. Circulating endothelial cells are proposed to be a noninvasive marker for the assessment of angiogenesis. The aim of this work was to evaluate, for the first time, the number of circulating endothelial progenitor cells (CEPCs) in the peripheral blood in childhood acute lymphoblastic leukemia (ALL) as a prognostic marker.
Materials and methods We quantified the number of CEPCs by flow cytometry in 50 childhood ALL patients at the time of diagnosis and in 30 healthy controls.
Results We found, statistically, that the CEPC number was significantly higher in the patient group than in the control group. There was a significant association between a high CEPC number with a higher total leukocytic count, the high-risk patient group, and a poor response to therapy, but no statistically significant difference regarding the hemoglobin concentration, the platelet count, cytogenetic analysis, immunophenotyping, and the French-American-British classification.
Conclusion The number of CEPCs is higher in childhood ALL patients and significantly linked to a high-risk disease status and chemoresistance.
Keywords: acute lymphoblastic leukemia; circulating endothelial progenitor cells; prognosis
|How to cite this article:|
Labib HA, Siam AG. Circulating endothelial progenitor cells as a prognostic marker in childhood acute lymphoblastic leukemia. Egypt J Haematol 2014;39:139-42
|How to cite this URL:|
Labib HA, Siam AG. Circulating endothelial progenitor cells as a prognostic marker in childhood acute lymphoblastic leukemia. Egypt J Haematol [serial online] 2014 [cited 2020 Nov 30];39:139-42. Available from: http://www.ehj.eg.net/text.asp?2014/39/3/139/148242
| Introduction|| |
Angiogenesis is the formation of new capillaries from established blood vessels. It is an essential process in growth, development, and wound healing  . However, it provides malignant cells with a survival advantage over their normal counterparts and confers the potential of metastasis  .
In the past years, the concept of angiogenesis has evolved from a simple model of the formation of new blood vessels from the pre-existing vasculature. However, it is also well established now that tumors can acquire their vasculature by various mechanisms, including postnatal vasculogenesis, a process during which circulating bone marrow drive endothelial progenitor cells home to sites of neovascularization, where they differentiate into endothelial cells and contribute to angiogenesis  .
There are several established methods for studying angiogenesis such as tumor microvessel density, which is more precise but invasive, and angiogenesis-related factors. However, general data regarding these angiogenic factors, especially in acute lymphoblastic leukemia (ALL) patients, are limited and many studies show conflicting results. Therefore, measuring circulating endothelial cells and bone marrow-derived circulating endothelial progenitor cells (CEPCs) has the potential advantage of being relatively noninvasive and feasible to perform serially and accurately. Moreover, it represents the summation of all the effects of various proangiogenic and antiangiogenic factors , .
Circulating endothelial cells are of interest because they may be surrogate markers of severe damage to the endothelium, and thus play a role in the pathophysiology of cardiovascular and inflammatory diseases. More interest in CEPCs arises because of their potential as stem cells and thus providers of neovascularization and the repair of the existing damaged endothelium, especially in malignancy. However, there is evidence that the two cell types may be linked and have much in common  .
The aim of this work was to study the CEPC count in childhood ALL and compare their levels with age-matched and sex-matched healthy children to prove their role in tumorigenesis and correlate these findings with clinical, hematological data, and the response to therapy to evaluate it as a prognostic marker in childhood ALL.
| Materials and methods|| |
This study was carried out on 50 consecutive newly diagnosed children with ALL and 30 age-matched and sex-matched healthy children. All children were recruited from the Pediatric Medical Oncology Unit and the Clinical Pathology Department of Zagazig University Hospital in the time period between 2010 and 2012. Informed consent to participate in the study was obtained from the patients' parents. Blood samples were obtained for controls. Blood samples and bone marrow aspirates were obtained at the time of diagnosis before initial cytotoxic therapy for leukemic children.
Children with ALL were categorized as either standard or high-risk categories. The standard risk category consisted of children aged from 1 to 10 years, with an initial total leukocytic count (TLC) lower than 50 × 10 9 /l; the high-risk category included children 10 years and older, who had a TLC of 50 × 10 9 /l or higher at the time of diagnosis. Re-evaluation of the patients 14 days after the induction therapy was performed to assess their initial response. The Berlin Frankfurt Munster ALL protocol was used for patients' treatment in this study  . Treated children were followed up for 24 months after induction therapy. The study was approved by the institution Ethics Review Board.
Complete blood counts (Sysmex N21, Japan) and CEPC counts were performed for all participants. The CEPCs that coexpressed CD133 (130-080-801, PE monoclonal antibody; MACS, Minneapolis, MN 55413, USA) and vascular endothelial growth factor (VEGF) receptor 2 (FAB357F, LWS06, FITC monoclonal antibody; R&D Systems) were identified using flow cytometry. The percentage of positive cells was converted to the absolute number of positive cells/μl using the following formula: the percentage of positive cells×TLC/100.
Acute lymphocytic leukemia was diagnosed on the basis of full history taking, thorough clinical examination, chest radiography, TLC, and examination of bone marrow aspiration with Leishman-stained films. ALL was classified according to the French-American-British (FAB) classification and immunophenotyping.
Immunophenotyping was performed using a panel of monoclonal antibodies analyzed by flow cytometry (FACScan; Becton Dickinson BD, San Diego, California, USA).
Cytogenetic analysis was performed on peripheral blood and bone marrow samples by G-banding analysis.
Data were statistically described in terms of the median and range. Comparison of quantitative nonparametric variables between the two studied groups was performed using the Mann-Whitney U-test, and comparison of more than two groups was performed using the Kruskal-Wallis test. A probability value (P value) less than 0.05 was considered statistically significant. All statistical calculations were performed using SPSS version 15 for Microsoft Windows (Statistical Package for the Social Sciences; SPSS Inc., Chicago, Illinois, USA). Associations between potential predictors and the CEPC count were evaluated by Cox proportional hazards models.
| Results|| |
The age of the ALL children ranged from 1 to 16 years, with a median of 8 years; there were 31 male and 19 female patients. The age of the control group ranged from 1 to 16 years, with a median of 9 years; 17 participants were male and 13 were female. The median value of TLC in children with ALL was 116.5 × 10 3 /μl, and in control children was 6.4 × 10 3 /μl (P < 0.001). The median CEPC count in the control group was 3.9/μl (range 1.2-8.5/μl) and for ALL children was 106.5 (range 17.4-189.3/μl), which was highly significant (P < 0.001) ([Table 1]). There was no significant relationship between the CEPC count and age, sex, pallor, fever, bleeding tendency, lymph node enlargement, splenomegaly, or central nervous system manifestations (not shown).
|Table 1 Characteristics of children with acute lymphoblastic leukemia (n = 50) and control children (n = 30)|
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There was a significant increase in the CEPC count in the ALL group with a higher TLC compared with those with lower TLC (P = 0.013) and in the high-risk group compared with those at low risk at diagnosis (P = 0.031). However, there were no significant differences in the hemoglobin concentration (P = 0.731), the platelet count (P = 0.384), and cytogenetic analysis (P = 0.851) ([Table 2]).
|Table 2 The circulating endothelial progenitor cell count/μl with different laboratory investigations, the FAB classification, and immunophenotyping subgroups in children with acute lymphoblastic leukemia (n = 50)|
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There was no significant difference in the CEPC count in the patient group according to the FAB classification (P = 0.622) or the immunophenotype subgroups (P = 0.978) ([Table 2]).
After induction therapy, 38 children with ALL achieved complete remission, nine showed residual disease, and three died early during induction. Children with residual disease had a significantly higher CEPC count than those who achieved complete remission (P = 0.006). During the follow-up period, seven patients relapsed, whereas 31 continued in complete remission. Patients with relapse had a significantly higher basal CEPC count (median = 135.2/μl) compared with those who remained in complete remission (median = 85.4/μl) (P = 0.007) ([Table 3]).
|Table 3 Comparison of the median count of circulating endothelial progenitor/μl and the response to therapy among children with acute lymphoblastic leukemia (n = 50)|
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By Cox regression analysis, the factors that added significance to the model were the risk category of the patients, the response to the therapy, and the follow-up outcome ([Table 4]).
|Table 4 The Cox proportional hazard model analysis of potential factors influencing the number of circulating endothelial progenitor cells|
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| Discussion|| |
Angiogenesis is a crucial component in the growth and the metastatic spread of solid tumors. It is a multistep process regulated by the impact of competing influences between inhibitors and angiogenesis activators  .
In recent years, there has been an increasing interest in researches on circulating endothelial cells in cancer and vascular diseases. Preclinical studies indicate that their numbers, viability, and kinetics correlate well with angiogenesis assays and can be used to monitor antiangiogenic drug activity  .
Many studies have tried to find the link between CEPCs and angiogenesis, CEPCs are associated with high expression levels of several chemokines and secretion of angiogenic growth factors  . These cells, coexpressing CD34 and AC133, are functionally nonadherent endothelial cells that have the capacity to migrate and differentiate into mature adherent endothelial cells. VEGF and fibroblast growth factor-2 are two growth factors that have been shown to enhance the growth of angioblasts and promote angiogenesis  . Chang et al.  have provided evidence indicating that tumor blood vessels may be mosaics in which both endothelial and tumor cells form the luminal surface.
CEPCs can be defined as migratory endothelial cells with the capacity to circulate, proliferate, and differentiate into mature endothelial cells similar to those of embryonic angioblasts  .
VEGF receptor 1 and VEGF receptor 2 were expressed on CEPCs effectively, inducing the mobilization of these cell populations into the circulation; CEPC levels in the peripheral blood increase within 24 h after exogenous VEGF administration ,, .
To the best of our knowledge, a study on the CEPC count in children with ALL has not been published or reported previously; hence, we cannot compare our results with other studies. In the current study, we found that the CEPC count was statistically significantly higher in ALL children than in the control group. Comparable results have been reported by Wierzbowska et al.  in acute myeloid leukemia patients. There was a significant positive association between the CEPC, count with a higher TLC and the high-risk patient group. This was in accordance with Rigolin et al.  , but that study was also conducted on acute myeloid leukemia patients.
CEPCs were detected more frequently in the peripheral blood and in the tumors of patients with a more invasive stage of disease  or in recurrence  , indicating a possible involvement in tumor progression and metastasis. We found a significant difference according to the response to therapy; a higher CEPC count was associated with a poor response to induction therapy and a higher rate of relapse. Many studies revealed that the CEPC level determines the sensitivity of tumors to both antiangiogenic and standard chemotherapy , .
One of the greatest hopes for the study of CEPCs is their potential use in cancer therapy as cellular vehicles for delivering suicide genes, toxins, or antiangiogenic molecules. We conclude that the CEPC count is higher in childhood ALL patients and its level is associated with a higher TLC, a poor response to therapy, and a higher relapse rate.
| Acknowledgements|| |
Conflicts of interest
There are no conflicts of interest.
| References|| |
Arnold F, West DC. Angiogenesis in wound healing. Pharmacol Ther
Leung DW, Cachianes G, Kuang WJ, Goeddel DV, Ferrara N Vascular endothelial growth factor is a secreted angiogenic mitogen. Science
Ria R, Piccoli C, Cirulli T, Falzetti F, Mangialardi G, Guidolin D, et al
. Endothelial differentiation of hematopoietic stem and progenitor cells from patients with multiple myeloma. Clin Cancer Res
Go RS, Jobe DA, Asp KE, Callister SM, Mathiason MA, Meyer LA, et al
. Circulating endothelial cells in patients with chronic lymphocytic leukemia. Ann Hematol
Blann AD, Pretorius A. Circulating endothelial cells and endothelial progenitor cells: two sides of the same coin, or two different coins?. Atherosclerosis
Flohr T, Schrauder A, Cazzaniga G, Panzer-Grümayer R, van der Velden V, Fischer S, et al
.International BFM Study Group (I-BFM-SG) Minimal residual disease-directed risk stratification using real-time quantitative PCR analysis of immunoglobulin and T-cell receptor gene rearrangements in the international multicenter trial AIEOP-BFM ALL 2000 for childhood acute lymphoblastic leukemia. Leukemia
Mangi MH, Newland AC. Angiogenesis and angiogenic mediators in haematological malignancies. Br J Haematol
Bertolini F, Shaked Y, Mancuso P, Kerbel RS The multifaceted circulating endothelial cell in cancer: towards marker and target identification. Nat Rev Cancer
Rehman J, Li J, Orschell CM, March KL Peripheral blood 'endothelial progenitor cells' are derived from monocyte/macrophages and secrete angiogenic growth factors. Circulation
Peichev M, Naiyer AJ, Pereira D, Zhu Z, Lane WJ, Williams M, et al
. Expression of VEGFR-2 and AC133 by circulating human CD34(+) cells identifies a population of functional endothelial precursors. Blood
Chang YS, di Tomaso E, McDonald DM, Jones R, Jain RK, Munn LL Mosaic blood vessels in tumors: frequency of cancer cells in contact with flowing blood. Proc Natl Acad Sci USA
Beerepoot LV, Mehra N, Vermaat JS, Zonnenberg BA, Gebbink MF, Voest EE Increased levels of viable circulating endothelial cells are an indicator of progressive disease in cancer patients. Ann Oncol
Rafii S, Heissig B, Hattori K. Efficient mobilization and recruitment of marrow-derived endothelial and hematopoietic stem cells by adenoviral vectors expressing angiogenic factors. Gene Ther
Asahara T, Takahashi T, Masuda H, Kalka C, Chen D, Iwaguro H, et al
. VEGF contributes to postnatal neovascularization by mobilizing bone marrow-derived endothelial progenitor cells. EMBO J
Ho JW, Pang RW, Lau C, Sun CK, Yu WC, Fan ST, Poon RT Significance of circulating endothelial progenitor cells in hepatocellular carcinoma. Hepatology
Wierzbowska A, Robak T, Krawczyñska A, Wrzesieñ-Ku? A, Pluta A, Cebula B, Smolewski P Circulating endothelial cells in patients with acute myeloid leukemia. Eur J Haematol
Rigolin GM, Mauro E, Ciccone M, Fraulini C, Sofritti O, Castoldi G, Cuneo A Neoplastic circulating endothelial-like cells in patients with acute myeloid leukaemia. Eur J Haematol
Naik RP, Jin D, Chuang E, Gold EG, Tousimis EA, Moore AL, et al
. Circulating endothelial progenitor cells correlate to stage in patients with invasive breast cancer. Breast Cancer Res Treat
Lin EH, Hassan M, Li Y, Zhao H, Nooka A, Sorenson E, et al
. Elevated circulating endothelial progenitor marker CD133 messenger RNA levels predict colon cancer recurrence. Cancer
Shaked Y, Henke E, Roodhart JM, Mancuso P, Langenberg MH, Colleoni M, et al
. Rapid chemotherapy-induced acute endothelial progenitor cell mobilization: implications for antiangiogenic drugs as chemosensitizing agents. Cancer Cell
Mancuso P, Colleoni M, Calleri A, Orlando L, Maisonneuve P, Pruneri G, et al
. Circulating endothelial-cell kinetics and viability predict survival in breast cancer patients receiving metronomic chemotherapy. Blood
[Table 1], [Table 2], [Table 3], [Table 4]