|Year : 2014 | Volume
| Issue : 4 | Page : 151-155
Study of CD123 (interleukin-3 receptor alpha Chain) and nuclear factor kappa B in adult acute myeloid leukemia patients
Inas Asfour1, Soha Yoyssef2, Maryse Ayoub1, Nevine Moustafa1, Hani Ayash1, Tussneem Elhassan3, Ghada M ElGohary1
1 Department of Internal Medicine, ADULT HSCT Program, Ain Shams University Hospitals, Cairo, Egypt
2 Department of Clinical Pathology, Ain Shams University Hospitals, Cairo, Egypt
3 Department of Oncology, Research Unit, King Faisal Specialist Hospital and Research Center, Riyadh 11211, Saudi Arabia
|Date of Web Publication||13-Dec-2014|
Dr. Ghada M ElGohary
Assisstant professor of Adult Hematology/BMT, Ain Shams University Hospitals, P.O. Box 1156, Cairo
Source of Support: None, Conflict of Interest: None
Background: The genomic alterations in acute myeloid leukemia (AML) affect the function of signaling molecules, transcription factors, and growth factor receptors besides they influence the response to treatment. Interleukin-3 receptor alpha chain (CD123) is overexpressed in about 45% of AML, this phenomenon was associated with high blast cell counts at diagnosis, with a worse prognosis. The transcription nuclear factor kappa B (NF-κB) can intervene in oncogenesis through its capacity to regulate the expression of a large number of genes that regulate apoptosis, cell proliferation, and differentiation. This study aimed to assess the relationship between CD123 expression and NF-κB level, in adult patients with AML at presentation and after induction therapy together with a well-known diagnostic and prognostic factors in AML. The study was conducted on forty adult newly diagnosed AML (group A) as well as twenty adult subjects who were hemato-oncologically free, as a control group (group B). Bone marrow (BM) examination and immunophenotyping by flow cytometry (FCM) on BM aspirate, for CD123 and quantitation of NF-κB in BM samples using ELISA (at three levels: Plasma, cytoplasmic and nuclear fraction of blasts, accordingly cytoplasmic/nuclear ratio) were performed for all studied subjects. In addition, levels of CD123 on blast cells and NF-κB were evaluated at D28 for remitted and resistant cases. All patients were positive for CD123 by FCM on BM blasts at time of diagnosis. The median cell counts expressing CD123 was 52.9 (34.4-75.6). Positive expression of CD123 was ≥20%, while in the control subjects CD123 expression was 25.1 (16.9-59.3), with significant decrease in CD123 expression after therapy, Besides, CD123 was also significantly higher in resistant patients compared to remitted patients (P = 0.009). As regard NF-κB, it was significantly higher in AML patients compared to control subjects at the three levels (plasma - cytoplasmic fraction - nuclear fraction) with a significant decrease after therapy. It was found that those NF-κB levels detected at follow-up (D28) on BM expression were significantly higher in resistant patients compared to remitted patients. While only The follow-up results, showed a highly significant positive correlation between CD123 expression on blast cells level and NF-κB plasma level alone (r = 0.587, P = 0.007) while there was not any correlation between CD123 and other two NF-κB levels neither at diagnosis nor at the follow-up. Conclusion: These data suggest that the correlation between CD123 and NF-κB was identified in variable results, and further elucidation of this role is likely to have important implications.
Keywords: Acute myeloid leukemia, CD123, nuclear factor kappa B
|How to cite this article:|
Asfour I, Yoyssef S, Ayoub M, Moustafa N, Ayash H, Elhassan T, ElGohary GM. Study of CD123 (interleukin-3 receptor alpha Chain) and nuclear factor kappa B in adult acute myeloid leukemia patients. J Appl Hematol 2014;5:151-5
|How to cite this URL:|
Asfour I, Yoyssef S, Ayoub M, Moustafa N, Ayash H, Elhassan T, ElGohary GM. Study of CD123 (interleukin-3 receptor alpha Chain) and nuclear factor kappa B in adult acute myeloid leukemia patients. J Appl Hematol [serial online] 2014 [cited 2020 Apr 2];5:151-5. Available from: http://www.jahjournal.org/text.asp?2014/5/4/151/146950
| Introduction|| |
Acute myeloid leukemia (AML) is a genetically heterogeneous clonal disorder characterized by the accumulation of acquired somatic genetic alterations in hematopoietic progenitor cells that alter normal mechanisms of self-renewal, proliferation and differentiation.  The genomic alterations in AML affect the function of signaling molecules, transcription factors, and growth-factor receptors and also determine the phenotype of the leukemia and influence the response to treatment. Moreover, the multiplicity of genomic changes that frequently coexist in a single leukemic cell reflects the successive transforming events that accumulate in the leukemic clone during the development of AML.  Interleukin-3 receptor alpha chain (CD123) is over expressed in about 45% of acute myeloid leukemia's , this phenomenon was associated with high blast cell counts at diagnosis, high rate of cycling of leukemic blasts and with a worse prognosis.  The transcription nuclear factor kappa B (NF-κB) can intervene in oncogenesis through its capacity to regulate the expression of a large number of genes that regulate apoptosis, cell proliferation and differentiation as well as inflammation, angiogenesis and tumor migration.  Constitutive or aberrant activation of NF-κB is frequently encountered in many human tumors as well as hematological malignancies including acute myeloid leukemia and is associated with a resistant phenotype and poor prognosis. In AML the mechanism of such persistent NF-κB activation is not clear but may involve defects in signaling pathways, mutations, or chromosomal rearrangements. Suppression of constitutive NF-κB activation inhibits the oncogenic potential of transformed Leukemic stem cells (LSC) and thus makes NF-κB an interesting new therapeutic target. 
| Subjects and Methods|| |
This study was conducted on 60 adult subjects attending the clinical hematology and oncology department, Ain Shams University hospitals through a period of 2 years from March 2009 to March 2011.
All subjects enrolled in this study signed an informed consent after the receiving approval of the experimental protocol by a local human ethics committee and the institutional review board. They were divided into 2 groups; (Group A): Included 40 patients with newly diagnosed AML, at presentation and after induction remission therapy (Group B) included 20 adult subjects who were hemato-oncologically free, as a control group.
They were all subjected to full history taking, thorough physical examination, laboratory investigations which included complete blood count and differentiation, blood chemistry, radiological investigations, as well as bone marrow (BM) examination for morphological assessment. Immunophenotyping and cytogenetic studies were performed for Group A patients only while for Group B, BM examination was indicated to confirm the diagnosis of patients' hematological condition.
One ml of BM was obtained via BM aspiration under complete aseptic conditions and dispensed into 2 sterile vacutainers containing Ethylene Diamine Tetra Acetic acid anticoagulant. One was used to obtain a cellular fraction and a plasma fraction to be stored at −70°C until assay of NF-κB, while the other was used for the flow cytometry (FCM) analysis of CD123 expression by blast cells.
Assessments of CD123 and nuclear factor kappa B
Immunophenotyping by FCM on BM aspirate, for CD123 and quantitation of NF-κB in BM samples using ELISA (at three levels: Plasma, cytoplasmic, and nuclear fraction of blasts, accordingly cytoplasmic/nuclear [C/N] ratio was calculated) were performed for all studied subjects.
Interpretation of results
The median count of cells expressing CD123 was obtained for each test and control specimen, and positive analysis was set at ≥20% of cells expressing CD123. The minimum detectable dose of human NF-κB is typically <15 pg/ml. The sensitivity of this assay, or lower limit of detection was defined as the lowest protein concentration that could be differentiated from zero which was in our case 47 pg/ml.
Standard computer program SPSS for Windows, release 10.0 (SPSS Inc., USA) was used for data entry and analysis. Continuous data were summarized using mean and standard deviation or median with Interquartile Range depending on the distribution of data. Categorical data were summarized using frequencies and proportions. Comparison of different variables in various groups was done using Student's t-test and Mann-Whitney test for parametric and nonparametric variables, respectively. For all tests a probability P < 0.05 was considered significant and < 0.001 was considered highly significant.
| Results|| |
In this study, all patients were positive for CD123 by FCM on BM blasts at time of diagnosis. The percent of cells expressing CD123 was 52.9 (34.4-75.6) in group A and 25.1 (16.9-59.3) in group B. The positive expression of CD123 was ≥20%. The median cell counts for NF-κB plasma levels in group A and B were 650 (510.5-1187.5) and 705 (465.0-827.5). For cytoplasmic fraction, the median cell count was 1410 (919-3318.8) and 910 (405-1115) for group A and B, respectively. Median cell count for nuclear faction was 1284 (839.3-1867.5) and 700 (540-915) for group A and B, respectively. The C/N ratio was 1.06 (0.76-2.11) in group A while 0.91 (0.85-1.3) in group B [Table 1].
|Table 1: Descriptive data of CD123 and NF-κB values for all studied patients (group A) and control group (group B) at diagnosis|
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At day 28 after induction therapy, BM examination was repeated for 20 patients (who lived) to assess BM remission. Twelve patients (12/20) were remitted (BM blasts <5%) and eight patients (8/20) were resistant (BM blast >25%). We found in our study that there was a significant decrease in CD123 expression and NF-κB levels after therapy. The level of CD123 expression decrease from 52.9 (34.4-75.6) to 25.1 (16.9-59.3).The levels of NF-κB detection decreased in: Plasma from 650 (510.5-1187.5) to 705.0 (465.0-827.5) - in cytoplasmic fraction from 1410.0 (919-3318.8) to 910 (405.0-1115.0)-in nuclear fraction from 1284 (839.3-867.5) to 700 (540, 915.0). The C/N ratio was 1.06 [0.76-2.11] in remitted patients, while it was 0.91 [0.85-1.30] in resistant patients [Table 2].
|Table 2: Descriptive data of CD123 and NF-κB values for surviving patients at D28 (n=20)|
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Regarding prognosis, CD123 expression was assessed, and it was significantly higher in resistant patients compared to remitted patients 21.4 (15.9-27.4) versus 64.4 (32.5-78.3), (P = 0.009). A significantly higher levels of NF-κB in plasma, cytoplasmic fraction, and nuclear fraction were obtained in the resistant patients' group compared to the remitted patients' group at follow-up (P = 0.021, P = 0.031, and P = 0.006, respectively, [Table 3].
|Table 3: Comparison between remitted versus resistant patients at follow-up|
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As regard the relation between CD123 and NF-κB, in our study, we observed that at follow-up there is a highly significant positive correlation between CD123 expression on blast cells level and NF-κB plasma level (r = 0.587, P = 0.007) which was not present at diagnosis. Furthermore, we did not found any further correlation between CD123 and other NF-κB expression parameters (cytoplasmic fraction-nuclear fraction-C/N ratio) at diagnosis or at follow-up.
| Discussion|| |
In this study, it was found that there is high level of CD123 expression on blast cell among all patients at diagnosis with median of 52.9 (34.4-75.6) which is going in agreement with Riccioni et al., 2011  and Muñoz et al., 2001.  Riccioni et al., 2011  investigated Immunophenotypic features of AML patients exhibiting high FLT3 expression not associated with mutations in 94 de novo cases of AML and found that in all these patients high expression of CD123 was present. Munoz et al., 2007 also consolidated our findings, as they analyzed the expression of the IL-3 receptor α chain (CD123) in 22 normal samples and in a wide panel of hematologic malignancies using FCM including in 64 patients with acute leukemia, 45 with AML and 19 with acute lymphocytic leukemia (ALL) 13 B-cell lineage ALL and 6 T-cell lineage ALL and found that all the AML cases except two patients with M7 and all the B-cell lineage ALL patients were CD123 positive. In contrast, all the T-cell lineage ALL cases tested were CD123 negative as well as in the control BM from healthy volunteers, CD123 was expressed in 0.27% (0.1- 0.6%) which was also in agreement with this study as CD123 expression in the control group (n = 20) was (12.3% ± 8.1%). Testa et al. 2004,  found that there is a good correlation between CD123 levels and the number of leukemia blasts at diagnosis, suggesting a role for CD123 in leukemic blast proliferation/survival. Furthermore, patients with elevated levels of IL-3R on their leukemic blasts exhibited a reduced number of complete remissions, higher relapse rate, and a lower survival rate than patients with normal/low CD123 levels. This was noted in our study as we found the level of CD123 expression on blast cell in resistant patients at follow-up was significantly higher than in remitted patients 64.4 (32.5-78.3) versus 21.4 (15.9-7.4) with P = 0.009 [Table 3] which supported the hypothesis that CD123 expression is correlated with bulk of leukemic blasts. On the other hand, several studies including Bueso-Ramos et al., 2004,  Baumgartner et al., 2002,  Wuchter et al., 2001  and Guzman et al., 2001  studied the role of NF-κB in AML. Baumgartner et al., 2002  studied NF-κB in myeloid blasts in 35 de novo AML patients with different AML subtypes (M0-M5). In agreement with this study, they found that in all of the studied different subtypes of AML samples, a marked activation of NF-κB was found, when compared to the controls, in both de novo newly diagnosed AML as well as relapsed cases and these results support the pivotal role of NF-κB pathway activation in AML. Also in line with our findings Bueso-Ramos et al., 2004,  Wuchter et al., 2001  and Guzman et al., 2001  found a marked activation of NF-κB in their studied patients with AML. Of particular importance to our hypothesis is trying to find if there is relation between NF-κB and white blood cell (WBC) count, both of Reilly et al., 2011  and Hewamana et al., 2008  have investigated this hypothesis and have supported our findings that there is a significant positive correlation between WBC and NF-κB (cytoplasmic fraction-nuclear fraction) at follow-up (r = 0.503, P = 0.024 and r = 0.650, P = 0.002, respectively. Regarding prognostic impact of NF-κB, in our study, we found a highly significant positive correlation between both PB and BM Blasts with NF-κB levels (cytoplasmic fraction-nuclear fraction) at follow-up and they were higher in resistant patients before and after therapy (r = 0.558 [P = 0.01], r = 0.734, [P < 0.001], r = 0.536 [P = 0.015], r = 0.704 [P = 0.001] respectively) which was in agreement with Page 7 of 10 http://www.mc.manuscriptcentral.com/jahem Journal of Applied Hematology Hewamana et al., 2008.  Furthermore Yeh et al., 2010  have investigated the role of NF-κB in the carcinogenesis of upper urinary tract urothelial carcinoma and it, s prognostic value for survival at follow-up and they noted that NF-κB may serve as a useful independent molecular marker to predict the outcome. Furthermore, to consolidate the prognostic significance of NF-κB, Izzo et al., 2006  have investigated the pretherapy and/or posttherapy cancer specimens (esophageal carcinoma) and were examined for activated NF-κB to be correlated with pathologic response to chemoradiation, metastatic potential, overall survival, disease-free survival, and type of chemotherapy or sequence used. Izzo et al., 2006  showed that pretreatment-activated NF-κB significantly correlates with clinical biology of esophageal cancer, and most importantly, with pathological response. Interestingly, Hafez et al., 2007,  studied markers of apoptosis and proliferation-related gene products including NF-κB as predictors of treatment outcome in childhood ALL. Their study was performed on 34 children with ALL and 39 healthy children as a control group. Apoptosis was assessed by cell morphology; DNA fragmentation; ELISA and Reverse transcription polymerase chain reaction for CD95, CD95 L, BcL-2, and NF-κB and FCM for CD95, CD40, CD49d, CD11a, and found that antiapoptotic factors: CD40, BcL-2, and NF-κB were all found to be higher in cases than controls and in refractory cases than those who achieved remission which was also in concordance with our findings. As regards the relation between CD123 and NF-κB, one of the studies held by Cavanagh et al., 2005  trying to determine whether CD123 + cells in the synovial tissue of rheumatoid arthritis patients were also expressing NF-κB. Their results have revealed that no CD123 + cells had expressed NF-κB although some expressed only the cytoplasmic level of NF-κB. This was in accordance to our findings that CD123 have only correlation with a plasma level of NF-κB and not with other parameters. In parallel, Guzman et al., 2001  analyzed NF-κB inprimitive AML cells by using CD34+/CD38−/CD123 as markers of stem BM cells and CD123 as marker for AML, and found that an identical pattern of NF-κB binding was observed for 3 of 3 CD34+/CD38−/CD123 + AML specimens which also indicates that NF-κB is relevant to the biology of AML stem cells, which furthermore consolidate our study findings. But on the other side, in keeping with the hypothesis that CD123 has or not correlation with NF-κB, two studies by Thompson et al., 2002  and Fohrer et al., 2004  in differentiated dendritic cells found no correlation between CD123 and NF-κB.
| Conclusion|| |
In our study, the results have expressed a positive correlation between CD123 expression on blast cell level and NF-κB plasma level only at follow-up and this correlation was not existed at diagnosis, While there is no further correlation between CD123 and other NF-κB expression parameters (cytoplasmic fraction-nuclear fraction-C/N ratio) at diagnosis or at follow-up, therefore, the relation between CD123 and NF-κB in de novo adult AML needs further larger studies.
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[Table 1], [Table 2], [Table 3]