|Year : 2013 | Volume
| Issue : 1 | Page : 7-12
Prognostic significance of flow cytometric quantification of circulating endothelial cells in chronic lymphocytic leukemia
Nihal M. Heiba1, Deena Samir Eissa1, Shereen A. El-Shazly2
1 Department of Clinical Pathology, Hematology Unit, Faculty of Medicine, Ain Shams University, Cairo, Egypt
2 Department of Internal Medicine, Hematology Unit, Faculty of Medicine, Ain Shams University, Cairo, Egypt
|Date of Submission||01-Oct-2012|
|Date of Acceptance||15-Oct-2012|
|Date of Web Publication||20-Jun-2014|
Deena Samir Eissa
Department of Clinical Pathology, Ain Shams University Hospitals, Ramses St, Abbasia, 11566 Cairo
Source of Support: None, Conflict of Interest: None
Accumulating evidences have supported the role of angiogenesis in the pathogenesis and progression of chronic lymphocytic leukemia (CLL). Detection of peripheral blood circulating endothelial cells (CECs) by flow cytometry is proposed to be a noninvasive indirect marker of angiogenesis. This work aimed to quantify CECs and endothelial progenitor cells (EPCs) by flow cytometry in patients with newly diagnosed CLL compared with healthy individuals, study their relationship with established risk predictors of the disease, and assess their prognostic significance.
Materials and methods
Flow cytometric quantification of CECs and EPCs was carried out for 50 newly diagnosed B-CLL patients and 20 healthy controls. Patients were followed up for assessment of the time to first treatment, response to therapy, and disease outcome.
Patients with CLL had higher counts of CECs (median, 24.6×106/l) and EPCs (median, 22.7×106/l) compared with the controls (median, 2.8 and 1.9×106/l for CECs and EPCs, respectively; P<0.001). CLL patients were subdivided according to the median values of CECs and EPCs into high and low CEC and EPC subgroups. Although high levels of CECs and EPCs were not related to established risk predictors of CLL (P>0.05), they were significantly related to higher white blood cell counts (P<0.001), shorter time to first treatment, and poor response to therapy (P<0.05).
This study shows that flow cytometric detection of peripheral blood CECs is a feasible indicator of abnormal angiogenesis in CLL that might be utilized as a biologic prognostic marker of a more aggressive disease course with a poor clinical outcome.
Keywords: angiogenesis, chronic lymphocytic leukemia, circulating endothelial cells, flow cytometry, prognosis
|How to cite this article:|
Heiba NM, Eissa DS, El-Shazly SA. Prognostic significance of flow cytometric quantification of circulating endothelial cells in chronic lymphocytic leukemia. Egypt J Haematol 2013;38:7-12
|How to cite this URL:|
Heiba NM, Eissa DS, El-Shazly SA. Prognostic significance of flow cytometric quantification of circulating endothelial cells in chronic lymphocytic leukemia. Egypt J Haematol [serial online] 2013 [cited 2020 Mar 31];38:7-12. Available from: http://www.ehj.eg.net/text.asp?2013/38/1/7/134796
| Introduction|| |
Angiogenesis is the development of new blood vessels from pre-existing vasculature, whereas vasculoneogenesis (or neoangiogenesis) is the de-novo formation of blood vessels 1. The induction and rate of angiogenesis depend on the balance between two functionally opposing groups of cytokines called angiogenic and angiostatic factors 2, as well as cell-to-cell and cell-to-extracellular matrix interactions. Circulating endothelial cells (CECs) are believed to act as building materials in regions with enhanced angiogenesis 3. These CECs, whether mature (in their resting or activated forms) or immature endothelial progenitor cells (EPCs), are extremely rare in normal peripheral blood (PB), representing between 0.01 and 0.001% of peripheral mononuclear cells 4. Angiogenesis has been shown to be crucial for tumor growth and dissemination 5, where new blood vessels are formed by CECs derived from pre-existing vessels and EPCs mobilized from the bone marrow (BM) 6.
Recently, there has been a growing interest to examine the role of CECs in various pathologic conditions including hematologic neoplasms 7–10; however, their role in tumor angiogenesis remains controversial 11,12. In chronic lymphocytic leukemia (CLL), considerable experimental and clinical data suggest the importance of angiogenesis in disease pathogenesis and progression, supported by the neovascularization of the BM and lymph nodes 13–15 and the prognostic relevance of several biologic markers of angiogenesis 16–18. The angiogenic microenvironment in CLL is supported by interactions between at least three subsets of BM cells producing angiogenic factors: the endothelial cells (ECs), malignant CLL cells, and stromal cells. When ECs are exposed to such angiogenic mitogens, they increase the expression of mRNA of various hematopoietic growth factors that produce an autocrine/paracrine effect on them, leading to their proliferation and migration to be incorporated into the vascular bed 13. However, contradictory or equivocal data have been obtained in CLL studies 19–21, supporting the fact that the degree of angiogenesis varies among patients and/or may depend on the method by which angiogenesis is measured.
Angiogenesis can be measured directly, by assessment of microvessel density, or indirectly, by measuring the levels of angiogenic or angiostatic factors. Moreover, evaluation of the number of PB CECs by flow cytometry (FCM) is a relatively noninvasive method to indirectly estimate angiogenesis 13. Different cell-surface markers have been utilized to detect CECs and EPCs using several approaches; however, consensus immunophenotypes have not been established. S-endo-1 (CD146) is a key marker in the identification of ECs, with its distribution being limited to ECs, smooth muscle cells, follicular dendritic cells, melanoma cells, and a subpopulation of activated T cells 4,22. Furthermore, delineation of mature CECs from immature EPCs was made possible with the discovery of CD133, an antigen that identifies primitive stem cells 4.
The aim of this work was to quantify CECs and EPCs by FCM in patients with newly diagnosed CLL compared with healthy individuals, study their relationship with established risk predictors of the disease, and assess their prognostic significance.
| Materials and methods|| |
This study was carried out on 50 newly diagnosed B-CLL patients attending the Hematology/Oncology Unit of Ain Shams University Hospitals. They were 31 men and 19 women, with a male to female ratio (M : F) of 1.6 : 1. Their ages ranged from 47 to 72 years (mean±SD, 58±12.1 years). Twenty age-matched and sex-matched healthy volunteers served as the control group: 32 men and 18 women (M : F, 1.7 : 1) ranging in age from 43 to 68 years (mean±SD, 54±10.6 years). Exclusion criteria in both groups were conditions known to increase the levels of CECs, such as poorly controlled diabetes mellitus, vascular diseases, chronic renal failure on dialysis, major surgery within 4 weeks, other clonal hematologic diseases, and the use of hematopoietic growth factors. Written informed consent was obtained from each patient and control before participation in the study. The study protocol was approved by the local ethical committee.
The diagnosis of B-CLL was made according to the National Cancer Institute-sponsored Working Group guidelines 23. Patients were subjected to (i) full history taking; (ii) thorough clinical examination with emphasis on pallor, purpuric eruptions, lymphadenopathy, and organomegaly; (iii) radiologic examination (radiographs, ultrasound, and computed tomography scan); and (iv) laboratory investigations, which included estimation of complete blood count using Coulter Cell Counter Gen-S (Beckman Coulter Inc., Fullerton, California, USA) with microscopic examination of PB smears, morphologic examination of BM aspiration/trephine biopsy smears, liver and kidney function tests, estimation of serum lactate dehydrogenase and serum β2-microglobulin levels, flow cytometric immunophenotyping of lymphocytes (Coulter Epics XL flow cytometer; Beckman Coulter Inc., Hialeah, Florida, USA) 24, conventional cytogenetic analysis by G-banding, and molecular cytogenetics using fluorescence in-situ hybridization probes (13q−, 17p−, 11q−, and +12; Vysis, Downers Grove, Illinois, USA) for conventional cytogenetic analysis-failed cases 25. Flow cytometric quantification of CECs and EPCs was carried out for all enrolled patients and controls. The main clinicopathologic characteristics of CLL patients are described in [Table 1].
|Table 1: Clinicopathologic characteristics of chronic lymphocytic leukemia patients|
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Patients were followed up over a period of 30 months (mean, 18 months; range 6–30 months). Time to first treatment (TFT) was calculated as the interval between diagnosis and the start of therapy (median TFT, 20 months). Treatment indication criteria were decided and patients’ response to therapy was evaluated according to the National Cancer Institute-sponsored Working Group guidelines 23. Intermittent chlorambucil administration was used as first-line therapy in the majority of cases. In young patients, treatment included administration of cyclophosphamide, vincristine, and prednisone, with or without anthracycline [C(H)OP regimens]. Fludarabine was used in refractory or relapsing patients and as front-line therapy in selected cases 24.
PB and BM samples were collected in potassium–ethylene diamine tetra-acetic acid (1.2 mg/ml), lithium heparin, and plain vacutainer tubes for morphologic, immunophenotypic, cytogenetic, molecular, and biochemical analyses.
Quantification of circulating endothelial cells and endothelial progenitor cells by flow cytometry
Quantification of CECs and EPCs was carried out by red cell lysis and the standard three-color staining flow cytometric technique within 3 h of blood collection (Coulter Epics XL flow cytometer; Beckman Coulter Inc.). The monoclonal antibody panel used was phycoerythrin cyanine 5-labeled anti-CD45 (Beckman Coulter Inc.), fluorescein isothiocyanate-labeled anti-CD146, and phycoerythrin-labeled anti-CD133 (Miltenyi Biotec, Bergisch Gladbach, Germany). Negative isotype-matched controls were used to determine nonspecific binding. At least 250 000 events were acquired for each sample, and analysis was carried out excluding the cellular debris in the side scatter/forward scatter dot plot. The gating strategy to detect CECs and EPCs was based on CD45 staining to exclude hematopoietic cells. CECs were identified as cells lacking CD45 expression, positive for CD146, and negative for CD133 (CD45–/CD146+/CD133–). EPCs were cells negative for CD45, coexpressing CD146 and CD133 (CD45–/CD146+/CD133+). The percentage of positive cells was converted into an absolute number (×106/l) using the formula [% of positive cells×white blood cell (WBC) count/100]×1000 10,26. A representative example of the flow cytometric quantification of CECs and EPCs in a CLL patient is shown in [Figure 1].
|Figure 1: A representative example of the flow cytometric quantification of circulating endothelial cells (CECs) and endothelial progenitor cells (EPCs) in a chronic lymphocytic leukemia patient. A gate was performed on CD45 staining to exclude hematopoietic cells (A). CECs were identified as CD45–/CD146+/CD133– (D). EPCs were identified as CD45–/CD146+/CD133+ (B).|
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Data were analyzed using SPSS version 14 (SPSS Inc., Chicago, Illinois, USA) on a Windows 7 operating system. Qualitative variables were expressed in the form of number and percentage; differences among groups were assessed using Fisher’s exact test. Quantitative variables were described in the form of mean±SD or median and range, as appropriate; comparisons between groups were made using Student’s t-test or the Mann–Whitney U-test for parametric and nonparametric data, respectively. Survival analysis was carried out according to the Kaplan–Meier method. Multivariate analysis using the Cox-proportional hazard regression model [hazard ratio (HR)] was carried out to identify significant independent prognostic factors on the basis of TFT. A P-value less than 0.05 was considered statistically significant.
| Results|| |
Quantification of circulating endothelial cells and endothelial progenitor cells
As shown in [Figure 2], the median of CECs in CLL patients was 24.6×106/l (range, 6.3–96.9), considerably exceeding the levels observed in healthy controls (median, 2.8×106/l; range, 2.3–16.1; P<0.001). The levels of EPCs in the PB of CLL patients followed the same pattern, being significantly higher (median, 22.7×106/l; range, 5.8–74.8) compared with the controls (median, 1.9×106/l; range, 1.4–12.5; P<0.001).
|Figure 2: Comparison between the median levels of circulating endothelial cells (CECs) and endothelial progenitor cells (EPCs) (×106/l) in chronic lymphocytic leukemia patients and controls (P<0.001).|
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On subdividing CLL patients according to the median CEC and EPC levels, 23 (46%) patients had high CEC levels and 27 (54%) had low CEC levels, whereas 21 (42%) and 29 (58%) had high and low EPC levels, respectively.
Circulating endothelial cell and endothelial progenitor cell levels in relation to clinicopathologic characteristics and established risk predictors
Patients with high CEC and EPC levels tend to have higher WBC counts than those with low CEC and EPC levels (P<0.001; [Figure 3]; however, they were comparable in terms of disease stage, CD38 expression, and cytogenetic-based risk classification (P>0.05; [Table 2].
|Figure 3: Mean white blood cell counts (×109/l) in relation to high and low circulating endothelial cell (CEC) and endothelial progenitor cell (EPC) levels in chronic lymphocytic leukemia patients (P<0.001).|
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|Table 2: Circulating endothelial cell and endothelial progenitor cell levels in relation to risk predictors and treatment of chronic lymphocytic leukemia|
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Circulating endothelial cell and endothelial progenitor cell levels in relation to chronic lymphocytic leukemia treatment and clinical outcome
On stratifying patients according to high and low CEC levels, 18/23 (78.3%) patients with high CEC levels and 11/27 (40.7%) patients with low CEC levels had to be started on therapy at some point during the study, the relationship being statistically significant (P<0.05). Similarly, the percentage of treated patients with high EPC levels (17/21; 81%) was significantly higher than that of treated patients with low EPC levels (12/29; 41.4%; P<0.05; [Table 2]. [Figure 4] shows a significantly shorter TFT in patients with high CEC levels.
|Figure 4: Kaplan–Meier curves of time to first treatment in chronic lymphocytic leukemia (CLL) patients according to circulating endothelial cell (CEC) levels.|
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As shown in [Table 3], the response to therapy also differed significantly among the patients according to the levels of CECs and EPCs. Thirteen of 18 (72.2%) treated patients with high CEC levels were nonresponders to treatment, with only 5/18 (27.8%) achieving complete remission (CR); however, 9/11 (81.8%) treated patients with low CEC levels achieved CR (P<0.05). In terms of EPCs, 12/17 (70.6%) treated patients with high EPC levels were nonresponders and only 5/17 (29.4%) achieved CR, whereas 9/12 (75%) patients with low EPC levels achieved CR (P<0.05).
|Table 3: Circulating endothelial cell and endothelial progenitor cell levels in relation to response to chronic lymphocytic leukemia treatment|
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Multivariate analysis of CLL TFT indicated high levels of CECs (HR, 2.3) and EPCs (HR, 2.1) to be independent prognostic factors of disease course and clinical outcome, along with disease stage, CD38 expression, and cytogenetic-based risk classification (P<0.05).
| Discussion|| |
CLL is a clinically heterogeneous disease characterized by a highly variable clinical course, with some patients progressing rapidly to death and others following a more stable nonprogressive course for many years. Cumulative survival data over the past decades have shown that patients with CLL are being diagnosed earlier in the course of their disease and living longer, leading to an increase in the variability of survival seen in CLL. In contrast to prevailing ideas, almost 70% of patients die from causes related to CLL. With the vast majority of treatments carrying significant toxicities, knowing whom and when to treat is extremely important. Therefore, the search for identifiable variables that can reliably and accurately predict prognosis might facilitate risk-adapted treatment strategies 27.
Several data suggest a role of angiogenesis in tumor growth and metastatic spread 2, 6, 9, 12. Although the direct methods for assessment of angiogenesis allow the quantification and morphologic evaluation of blood vessels, they have the disadvantage of being invasive, in addition to the inherent sampling error risk of not being representative of the entire neoplasm 13. Recently, quantification of CECs was utilized as an indirect measure of angiogenesis, with several reports pointing to their increase in patients with solid tumors and hematologic malignancies, for example, acute myeloid leukemia, multiple myeloma, and myelodysplastic syndromes 7–10. The role of angiogenesis in the pathophysiology of CLL showed contradictory results 14, 15, 17, 19, 21 and remains to be fully elucidated, which seems reasonable in view of the wide clinical heterogeneity of the disease.
Although the exact definition and a universally accepted consensus for immunophenotypic profiles of CECs and EPCs remain matters of debate 4, flow cytometric quantification of PB CECs and EPCs in the present study represented an easy-to-use indirect method for estimation of angiogenesis status, being a relatively noninvasive procedure, readily available in most modern laboratories. Moreover, the profile of CECs may represent a sum of all the effects of various positive and negative regulators (proangiogenic and antiangiogenic, inflammatory and hemostatic) on the endothelium 28.
In this study, levels of CECs and EPCs were significantly higher in CLL patients than in healthy controls. Similar results have been reported in a few reports 26, 28, 29 despite the fact that each of them utilizes a slightly different combination of flow cytometric markers.
Comparable with data obtained by Rigolin et al. 26, this study showed that the elevation in CEC and EPC levels in CLL patients was significantly associated with higher WBC counts but showed no relation to established risk predictors (Rai stage, CD38 expression, and cytogenetic markers). Interestingly, Letilovic et al. 13 explained the proangiogenic environment in CLL cells to be a reflection of the greater number of leukemic cells present, which would naturally produce larger amounts of angiogenic factors that lead to increased angiogenesis. Moreover, Xia et al. 30 reported that angiogenic mitogens act as prosurvival factors for CLL cells by activating antiapoptotic signaling pathways. However, Go et al. 28 failed to find any relationship between CEC levels and WBC counts. In addition, Gora-Tybor et al. 29 were able to relate the increase in the number of CECs with more advanced clinical disease stages. This variation in statistical observations is probably because of differences in the sample size and distribution of disease stages among the studied populations.
In the current work, the majority of CLL patients with high levels of CECs (78.3%) and EPCs (81%) showed a shorter TFT in relation to those with low levels of CECs and EPCs. In addition, most of the treated patients with high levels of CECs (72.2%) and EPCs (70.6%) were poor responders. Multivariate analysis of CLL TFT recognized high levels of CECs and EPCs as independent prognostic factors of CLL. Taken together, these data suggest that a subset of CLL patients with a more aggressive disease course (indicated by shorter TFT), poor response to therapy, and thereby a worse prognosis can be identified by the presence of high levels of CECs and EPCs in PB. Similar findings have been reported by Rigolin et al. 26. However, although Go et al. 28 found no such relation, probably because of the small sample size (20 patients) with earlier disease stages, of their four patients with high levels of CECs, three had progressive disease.
Overall, the aforementioned data confirm the increased levels of PB CECs and EPCs in CLL patients, with higher levels being associated with a more progressive disease of poorer prognosis and suggesting the relationship of CECs with the phenomenon called ‘angiogenic switch’, which characterizes the shift to a more aggressive course of the disease 26. These findings provide the rationale for investigating antiangiogenic agents in CLL, including new drugs such as lenalidomide, in which CEC levels can be used as a defining marker for patients eligible for this kind of therapy and as a monitor for determining their efficacy 13.
| Conclusion|| |
This study shows that flow cytometric estimation of CECs in patients with CLL is feasible and may aid evaluation of disease activity by defining a subset of patients with a more aggressive course. Further studies are warranted for the development of specifically ‘tailored’ treatment strategies targeting the angiogenic process in this group of patients and for determining the possibility of using CECs as a surrogate marker to monitor clinical response.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3]