|Year : 2019 | Volume
| Issue : 4 | Page : 208-217
Leukemia stem cell markers (CD 123 and CD25) are poor prognostic markers in patients with adult acute myeloid leukemia
Botheina A.T Farweez1, Azza E Hashem1, Walaa A Elsalakawy MD 2
1 Department of Clinical Pathology, Clinical Hematology and Bone Marrow Transplant Unit, Faculty of Medicine, Ain Shams University, Cairo, Egypt
2 Department of Internal Medicine, Clinical Hematology and Bone Marrow Transplant Unit, Faculty of Medicine, Ain Shams University, Cairo, Egypt
|Date of Submission||31-Aug-2019|
|Date of Acceptance||26-Oct-2019|
|Date of Web Publication||20-Jul-2020|
Walaa A Elsalakawy
Department of Internal medicine, Clinical Hematology and Bone Marrow Transplant Unit, Faculty of Medicine, Ain Shams University, Cairo, 11241
Source of Support: None, Conflict of Interest: None
Background Acute myeloid leukemia (AML) still remains a challenge for hematologists. Though an impressive number of prognostic factors have been identified in AML, it still ranks as one of the highest cancer-related deaths. Several studies have indicated its origin from a rare population of leukemic cells, known as leukemic stem cells, which initiate the disease and contribute to frequent relapses. Leucocyte interleukin-3 receptor α (CD123) and leukocyte interleukin-2 receptor α (CD25) are regarded as markers of leukemia stem cells.
Aim The aim of this study was to investigate CD123 and CD25 expressions in newly diagnosed patients with AML by flow cytometry and correlate their expression with disease prognostic parameters and patients’ outcome at day 28 of therapy.
Patients and methods This study was conducted on 30 newly diagnosed patients with AML admitted to Ain Shams University Hospitals. The expression of CD123 and CD25 was assessed, where gated blast cells were stained for CD45, CD38, CD34, CD123, and CD25.
Results In the current study, CD123 was expressed in 13/30 (43.3%) patients, and CD25 was expressed in 4/30 (13.3%) patients. CD123 expression positively correlated with higher total leukocytic count and bone marrow blast percentage and CD25 expression. Both CD123 and CD25 expression had a significantly poor effect on outcome even in the good prognostic cytogenetic subgroups.
Conclusion Results of our study clearly demonstrate the poor prognostic significance of CD123 and CD25 expression in patients with AML. This may represent an additional prognostic tool in risk-stratified management of patients with AML.
Keywords: acute myeloid leukemia, leukemia stem cell markers, prognosis
|How to cite this article:|
Farweez BA, Hashem AE, Elsalakawy WA. Leukemia stem cell markers (CD 123 and CD25) are poor prognostic markers in patients with adult acute myeloid leukemia. Egypt J Haematol 2019;44:208-17
|How to cite this URL:|
Farweez BA, Hashem AE, Elsalakawy WA. Leukemia stem cell markers (CD 123 and CD25) are poor prognostic markers in patients with adult acute myeloid leukemia. Egypt J Haematol [serial online] 2019 [cited 2020 Aug 12];44:208-17. Available from: http://www.ehj.eg.net/text.asp?2019/44/4/208/290232
| Introduction|| |
Acute myeloid leukemia (AML) originates from a special proportion of leukemia stem cells (LSC), which possess self-renewal capacity and are responsible for the continued growth and proliferation of the bulk of leukemia cells. It is believed that LSC are also the root cause for the treatment failure and relapse of AML because LSC are often resistant to chemotherapy .
LSC and hematopoietic stem cells (HSC) share similar CD34+ CD38- surface immunophenotype. The search of cell surface markers unique to LSC or at least differentially expressed has attracted intensive enthusiasm in hemato-oncology field. Such markers will provide excellent therapeutic windows for specifically targeting LSC, while sparing normal HSC, and are expected to be much tolerable for patients with AML .
CD123 is the alpha chain of interleukin-3 receptor (IL3-R), and CD25 is the alpha chain of the interleukin-2 receptor (IL-2R); they have been shown to be highly expressed on LSCs .
The objective of the present study was to investigate CD25 and CD123 expressions by flow cytometry in the diagnosis of adult Egyptian patients with AML and to explore their relationship with disease prognostic parameters and patients’ outcome at day 28 of therapy, with the aim to apply them in routine clinical practice.
| Patients and methods|| |
This study included 30 newly diagnosed adult patients with AML admitted to and followed up at the Clinical Hematology Unit of Internal Medicine Department, Ain Shams University Hospitals. Patients’ diagnosis, management, and follow-up were performed according to WHO classification  and European Society for Medical Oncology (ESMO) Clinical Practice Guidelines for diagnosis, treatment and follow-up of AML in adult patients . Patients’ characteristics were evaluated at diagnosis by history, physical examination, complete blood count using Coulter LH 750 analyzer (Miami, Florida, USA), examination of Leishman-stained peripheral blood, and bone marrow (BM) aspiration smears.
Routine diagnostic immunophenotyping of the BM aspirate was performed on EPICS XL coulter flow cytometer (6511 Bunker Lake Blvd. Ramsey, MN, USA) using a panel of monoclonal antibodies, including the following: B-cell markers such as CD10, CD19, and CD20; T-cell markers, such as CD2, CD3, CD5, and CD7; myeloid markers, such as CD13, CD33, CD15, and CD17; monocytic marker, such as CD14; common progenitor markers, such as CD34, HLA-DR, and CD38; and cytoplasmic markers, such as MPO, CD79a, and CD3.
Samples were considered positive for a certain marker when greater than or equal to 20% of cells were expressing it, except for CD34 where its expression by 10% of cells was sufficient to confer positivity .
Conventional karyotyping and fluorescence in-situ hybridization in selected cases were performed. Patients were followed up at the day 28 from the beginning of the induction therapy.
Informed consent was obtained from all participant individuals. The study was conducted in accordance with the stipulations of the Local Ethical and Scientific Committees of Ain Shams University, and the procedures respected the ethical standards in Helsinki declaration of 1964.
Flow cytometric assessment of CD123 and CD25
For each sample analyzed, in addition to the test tube, one control tube was required for isotopic matched controls. For a blood sample, optimal staining was obtained using a number of leukocytes between 5 and 10×103 cells/µl. If the leukocyte concentration was greater than 10×103 cells/µl, it was diluted with PBS (pH: 7.2±0.2).
50 µl of prepared sample containing at least 5×103 cells were added to each sample tube.
- 5 µl each of PE-conjugated anti-CD123 monoclonal antibody, FITC-conjugated anti-CD25 monoclonal antibody, PC7-conjugated anti-CD45, ECD-conjugated anti-CD38, and PC5-conjugated anti-CD34 (R&D Systems, 19 Barton LaneAbingdon Science Park, Abingdon, UK) was added to each sample tube.
- Tubes were incubated for 15–20 min at room temperature and protected from light. Overall, 1–2 ml of ammonium chloride-based erythrocyte lysing solution was added to every tube and incubated for 5–10 min. Tubes were vortexed, and then washed with PBS (pH: 7.2±0.2). Cells were suspended in 0.5 ml PBS and analyzed using Navios flow cytometer (Coulter Electronics, Indianapolis, IN, United States).
Analysis was performed using Navios software, where 10 000 events were analyzed per case. A five-color flow cytometric assay protocol was constructed in which CD45/SSC gating was used to locate immature cells, and then CD38-negative blasts were selectively gated. The expression of CD123 (cutoff 20%) , CD25 (cutoff 20%) (cutoff for positivity for CD25 was variable between studies; Terwijn et al.  and Cerny et al.  suggested using a cutoff ≥10% based on the maximal expression of CD25 on CD34-positive myeloid blasts in normal marrow, whereas Gönen et al. , used the cutoff level of 20% as it has been known as the standard, and we consider this as the best cutoff level to exclude false-positive results], and CD34 (cutoff 10%) were assessed in terms of percentage of expressing cells ([Figure 1]).
|Figure 1 Representative flow cytometry scatter diagrams for CD123/CD25/CD34 on CD38− blast cells in one of the studied acute myeloid leukemia cases.|
Click here to view
Statistical analysis was performed using SPSS V17 (Corporate headquarters1, New Orchard Road Armonk, New York, United States). Quantitative data were represented as mean and SD for parametric data and as median and range in nonparametric data. Qualitative data were represented as number and percentage.
Comparisons of qualitative variables were conducted between groups using theχ2, and comparisons of quantitative variables were conducted between groups using the Mann–Whitney U-test for nonparametric data and Student’s t-test for parametric data. However, comparisons between more than two groups with parametric distribution were done by using one-way analysis of variance and Kruskal–Wallis test for nonparametric distributions.
In addition, correlations between quantitative variables within groups were performed using the Pearson correlation coefficient. P less than 0.05 and less than 0.001 were set as statistically significant and highly significant, respectively.
| Results|| |
The studied patients included 13 (43.3%) males and 17 (56.6%) females, with male : female ratio of 1 : 1.3. Their age ranged from 20 to75 years, with a mean value of 44.00±16.88 years. They were classified according to FAB grouping, but none of the patients were diagnosed as M6 or M7.
Conventional cytogenetic analysis was performed, where three (10%) out of 30 patients showed failed mitosis and could not undergo cytogenetic analysis. The remaining 27 patients were divided into the following: 14 (51.9%) patients out of 27 had normal karyotype and the other 13 patients underwent fluorescence in-situ hybridization analysis. Patients were further classified as favorable, unfavorable, and intermediate prognostic groups according to cytogenetic analysis, where t (8;21) (q22;q22), inv16 (p13;q22), and t(15;17) (q22;q21) were considered as favorable prognosis, whereas normal karyotype was considered intermediate prognosis, and 11q23 rearrangement was considered as unfavorable prognosis . Expression of CD34, CD123, and CD25 was performed on the gated blast cells, which were CD38 negative. Studied laboratory data are shown in [Table 1].
Relation between CD123 and different parameters
There was a statistically significant association between CD123 expression and each of total leukocytic count (TLC) and BM blast cells percentage. Higher median value for TLC and BM blast cells % was found in CD123⁺ patients (P=0.002 and 0.013, respectively). Moreover, CD123 expression and FAB subtypes showed a statistical significant relation (P=0.039), where higher percentage (38.46%) of CD123⁺ expression was found among M4 FAB subtype. Moreover, there was a statistically significant association between CD25 expression and CD123 expression (P=0.006), where the four patients positive for CD25 were CD123 positive. However, there was no statistically significant relation between CD123 expression and either demographic, clinical data, or other studied laboratory parameters ([Table 2]).
Relation between CD123 and CD34 co-expression and different parameters
There was no statistically significant association regarding CD123/CD34 co-expression and any of the studied parameters.
Relation between CD25 and different parameters
There was no statistically significant association regarding CD25 expression and any of the studied parameters except for a statistical significant relation with CD123 expression ([Table 3]).
Outcome of patients with acute myeloid leukemia
On assessing patients’ outcome at day 28, 12 (40%) patients achieved hematological remission, whereas 10 (33.3%) patients did not achieve hematological remission, and eight (26.7%) patients died shortly after their diagnosis.
Relation of outcome and studied parameters:
The relationship between the outcome and FAB subtypes showed a statistical significant relation (P=0.048). Higher percentages of patients who did not achieve hematological remission at day 28 were found among M4 FAB subtype (40.0%) and higher percentages of patients who died were found among M5 FAB subtype (37.5%), whereas higher percentages of patients who achieved hematological remission at day 28 were found among M2 FAB subtype (50.0%) ([Table 4]).
There was no statistically significant relation between the outcome and different marker expression, where all CD25+ cases died shortly after diagnosis and they were CD123+ also. However, among CD123+ cases, 8 out of 13 did not achieve hematological remission and 5 of 13 died shortly after diagnosis ([Figure 2]). Moreover, it was of notice that eight patients out of the 10 patients who did not achieve hematological remission showed CD123 co-expression, and six of them showed CD123 and CD34 co-expression ([Table 4]); higher median value for CD123 expression and CD123/CD25 co-expression were found among patients who died shortly after diagnosis (P=0.012 and 0.048, respectively) ([Table 5]).
|Figure 2 (a) Relation between CD123 expression and the outcome of studied patients with acute myeloid leukemia; (b) relation between CD25 expression and the outcome of studied patients with acute myeloid leukemia.|
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There was a statistically significant relation between different cytogenetic aberrations and patient outcome at day 28 (P=0.033). Patients with normal karyotype were 14 patients (51.8% of total cytogenetically studied patients); they represented 75% of patients who died, 50% of patients who did not achieve hematological remission, and 36.4% who achieved hematological remission, but it was noticed that 27.3% of patients who achieved hematological remission presented with t(8;21) or t(15;17) (good prognostic cytogenetic group), and although inv16 is one of the good prognostic cytogenetic aberrations, three patients out of four with inv16 did not achieve hematological remission ([Table 4]), and those patients were found to have coexpression of CD123 and CD25.
CD34 had a statistically significant negative correlation with BM blasts (r: −0.405, P: 0.026), CD25 had a statistically significant positive correlation with CD123 (r=0.399, P=0.029), and CD123 had a statistically significant positive correlation with TLC and BM blasts (r=0.365 and 0.553 and P=0.048 and 0.002, respectively).
| Discussion|| |
AML is a clinically and biologically heterogeneous disease for which prognostic factors have become increasingly important for the choice of the appropriate therapy. New prognostic tools based on biological analyses which are accurate and easily available in clinical settings are needed .
LSC are reported to play a crucial role in the development and progression of many hematological malignancies, including AML . CD123 and CD25 have been reported as LSC markers; such markers will provide excellent therapeutic windows for specifically targeting LSC, while sparing normal HSC . The current study was conducted to evaluate the clinical value of investigating CD25 and CD123 expression at initial diagnosis as prognostic factors for AML via investigating their correlations with other well-established prognostic factors and patients’ outcome at day 28 of induction therapy.
In agreement with other previous studies ,,,, this study showed that CD123 was expressed on the blast cells in 43.3% of patients. There is some variation in the reported percentage of positivity for CD123 between these studies ranging from 40 to more than 90%, which could be explained by the different methodologies used to assess CD123 expression (flow cytometry vs immunohistochemistry), variation in number of studied patients, and variation of the cells on which CD123 expression was tested and the FAB subtypes as well. However, it could be concluded that CD123 is very frequently expressed on AML blasts and enforces previous reports about its role in AML development or pathology.
On exploring the relation between the expression of CD123 and studied laboratory data, CD123 was associated with higher TLC and higher percentage of BM blasts. This might suggest that the expression of CD123 probably offers a proliferation advantage to malignant cells and might explain the association with poor prognosis, as they are prognostic factors in themselves; similar results were documented by several previous studies ,,,.
The relation of CD123 to proliferation advantage has been emphasized by the study of Testa et al.  who sorted leukemic cells according to strong or low expression of CD123 and concluded that cells expressing CD123 displayed higher growth activity but lower differentiation ability, and exhibited increased resistance to apoptosis triggered by growth factor (IL3) deprivation.
The distribution of CD123 expression among the FAB subgroups showed the highest percentages of CD123 positivity observed in M4 FAB subtype, and none of the M1 FAB subtype was positive. Ehninger et al. , found a high percentage for CD123 positivity of 80–90% in M4, M4eos, M0, M1, and M5 and 100% of M3 and M6 leukemia, but they showed lower percentage for CD123 positivity in M2 FAB. However, several studies did not find a significant difference in CD123 expression and FAB grouping ,,. This might suggest that its prognostic impact is not confined to a specific group even in the subgroups of AML with favorable outcome.
A significant correlation between CD123 expression and CD25 expression was found where all CD25+ patients (4 patients) were CD123+. Similarly, Gönen et al. , performed gene expression profiling analysis in CD25+ intermediate-risk patients and observed increased expression of CD123 in this subgroup.
Upon grouping patients according to their cytogenetic prognostic groups, no significant difference in CD123 expression and the three cytogenetic prognostic groups was found, but it was of note that 6 of 11 (54.5%) patients positive for CD123 were in the intermediate prognostic groups. In agreement, Ehninger et al. , reported the same finding and suggested that generally patients might profit to the same extent from targeted therapies against CD123 and CD33, as only ∼4% of AMLs were negative for both marker.
Previous works done by Riccioni et al. , Rollins-Raval et al. , and Ehninger et al.  who studied the relation of CD123 and molecular aberrations, showed higher CD123 expression in FMS-like tyrosine kinase-internal tandem duplication (FLT3-ITD+) AMLs compared with FLT3 wild-type allele (FLT3-WT); thus, this enforces the poor prognostic effect of CD123 expression.
CD25 was positive in 13.3% of patients in the present study and showed a significant relation with CD123 expression but not with any other studied clinical and laboratory parameters. Likewise, Gönen et al.  and Cerny et al. , reported nearly same percentage of positivity. Meanwhile, Ikegawa et al.  and Gönen et al. , reported that CD25 expression was associated with higher TLC in addition to higher percentage of peripheral blood blast cells .
There was no significant relation between CD25 expression and the three cytogenetic prognostic groups, but it was noticed that 50% of CD25+ cases were found in the cytogenetically intermediate-risk AML (mostly with normal karyotype). Moreover, Terwijn et al. , Cerny et al. , and Gönen et al.  reported similar results. Additionally, they demonstrated that CD25+ patients with intermediate-risk cytogenetics have a greater likelihood of harboring unfavorable risk mutations compared with CD25−cytogenetically intermediate-risk patients. Thus, this highlights the great importance of CD25 expression especially in the cytogenetic intermediate-risk group patients.Previous work by Gönen et al.  had shown a high frequency of FLT3-ITD+ cases in CD25+ AML (76% of cases) and documented that CD25− FLT3-ITD+ patients fared equally well as CD25− FLT3-WT patients suggesting that lack of CD25 expression outweighs the well-known adverse prognostic effect of the FLT3-ITD mutation.
Regarding the patients’ outcome at day 28 after induction, poor outcome (death shortly after diagnosis or nonremission state) showed a highly significant relation with CD123 expression, significant relation with CD25 expression, and significant relation with CD123/CD25 co-expression and CD123/CD34 coexpression. This is in line with Testa et al.  who reported that patients with CD123 overexpression had a lower complete remission and survival duration. Moreover, Vergez et al.  reported that the number of CD34+/CD123+/CD− cells was predictive of AML patient outcome and a proportion of CD34+/CD123+/CD38− cells greater than 15% in patients with AML, and an unfavorable karyotype was associated with a lack of complete remission; furthermore, the presence of more than 1% of CD34+/CD123+/CD38− cells had a negative effect on disease-free survival and overall survival.
Similarly, Gönen et al.  reported that CD25 expression was associated with a reduced response to induction chemotherapy. In addition, several independent reports using diverse patient cohorts established a significant positive correlation between the percentage of the CD25 positive leukemic blast population and poor overall survival or relapse-free survival ,.
| Conclusion|| |
In conclusion, we suggest that CD25 and CD123 can be incorporated as additional biomarkers to improve prognostication in AML, and these patients should be considered for tailored immunotherapies targeting CD123 and CD25, which are likely to enhance treatment efficacy in patients with AML.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]