|Year : 2016 | Volume
| Issue : 4 | Page : 161-167
Value of human CLEC12A expression in acute myeloid leukemia
Gehan M Hamed MD , Mona F Abdel Fattah
Department of Clinical Pathology, Faculty of Medicine, Ain Shams University, Cairo, Egypt
|Date of Submission||24-Jun-2016|
|Date of Acceptance||22-Sep-2016|
|Date of Web Publication||20-Jan-2017|
Gehan M Hamed
Department of Clinical Pathology, Faculty of Medicine, Ain Shams University, 67 El Nasr Street,
Sheraton Heliopolis, Cairo, 11799
Source of Support: None, Conflict of Interest: None
Background The need for identification of new specific, stable antigens during the course of acute myeloid leukemia (AML) is warranted to improve diagnosis, relapse detection, and eradication of leukemic cells.
Aim We measured the surface marker human C-type lectin domain family 12, member A (CLEC12A), in 60 AML patients, 24 acute lymphoblastic leukemia (ALL) patients, and 20 controls by flow cytometry, to determine its diagnostic utility and stability in AML.
Results CLEC12A was positively expressed in all studied AML patients, negative expression was detected in ALL patients, and normal CD34+ cells of the controls with significantly higher mean % expression and median fluorescence intensity was detected among AML patients (P<0.001). Receiver operating characteristic curve analysis revealed that CLEC12A at a cutoff value of at least 15.1% can diagnose and differentiate between AML and ALL with 100% sensitivity and specificity. CLEC12A was found to be positively expressed both in children and adult AML patients with significantly higher mean % expression and median fluorescence intensity in children (P=0.046), and a significant difference was found between different French–American–British Classification subtypes (P<0.001). No significant difference was detected as regards sex, newly diagnosed untreated AML patients versus AML patients in relapse (79.1±11.7 vs. 67.4±19.3, P=0.052), CD34+ versus CD34− leukemic blast cells (75.1±20.7 vs. 74.1±21.7; P=0.899), or between different cytogenetic prognostic risk groups (P>0.05). The marker was found to be positively expressed without significant difference in paired diagnosis/relapse samples, indicating its stability during the course of disease and after treatment.
Conclusion CLEC12A is a specific and stable diagnostic marker of AML that could improve leukemia-associated immunophenotypes both in CD34+ and the poorly characterized CD34− patients by flow cytometry. In addition, low expression of CLEC12A on normal CD34+ progenitor cells positions the marker as a potential therapeutic target.
Keywords: acute myeloid leukemia, CLEC12A, diagnosis, stability
|How to cite this article:|
Hamed GM, Abdel Fattah MF. Value of human CLEC12A expression in acute myeloid leukemia. Egypt J Haematol 2016;41:161-7
| Introduction|| |
Acute myeloid leukemia (AML) is a common hematological malignancy, accounting for approximately 80% of acute leukemia in adults and 20% of acute leukemia in children . Timely and accurate diagnosis of hematologic malignancies is crucial to appropriate clinical management . Despite a high remission rate (approaching 80% in younger adults) after intensive chemotherapy, only 30–40% of patients survive 5 years after diagnosis . Many patients will experience relapse, which is caused by the presence and outgrowth of sustaining leukemic stem cells in the bone marrow (BM), termed minimal residual disease .
Flow cytometric immunphenotyping of leukemic cells has a crucial role in rapid identification of cell line, definition of the maturation stage, and finding possible aberrant antigens, which in turn serves for individual treatment monitoring and detection of residual disease ,. In the absence of leukemia-specific markers, the distinction between leukemic and normal immature cells relies on the expression of antigen combinations defining leukemia-associated immunopheotypes (LAIPs), which are absent or extremely infrequent in normal bone marrow ,. However, LAIPs are very different from patient to patient, and they are not necessarily stable over the course of disease . Consequently, new leukemia-associated antigens are highly warranted for contribution to diagnostic and prognostic information, improving relapse detection, and ideally eradication of LSCs through antibody-mediated targeted therapy.
Myeloid inhibitory C-type-like lectin (MICL), C-type lectin-like molecule 1 (CLL-1), or dendritic-cell-associated lectin 2 (DCAL-2) [official name: C-type lectin domain family 12, member A (CLEC12A)] has been identified independently by several groups ,,,. It is a type II transmembrane glycoprotein comprising an extracellular C-terminal lectin domain (CTLD), a stalk, transmembrane region, and an N-terminal cytoplasmic tail. It is expressed on a variety of cells including primarily granulocytes, monocytes, and perhaps some natural killer cells in normal peripheral blood and BM, macrophages, and dendritic cells, whereas it is absent in nonhematological tissues . This expression pattern implicates an important role of CLEC12A in hematopoiesis and potentially in myeloid leukemogenesis; there is some evidence that it is involved in the control of myeloid cell activation during inflammation . We aimed to determine the diagnostic utility of CLEC12A expression in differentiating AML from ALL and normal hematopoietic progenitors, and its stability after treatment in AML patients.
| Patients and methods|| |
This study was conducted on 60 AML patients (44 patients were newly diagnosed untreated AML patients and 16 patients were in relapse at different points of treatment), including eight (13.3%) children and 52 (86.7%) adults, in addition to 24 newly diagnosed ALL patients [included 12 (50%) children and 12 (50%) adults]. AML patients were treated for curative intent and were followed up for 12 months to detect their response to treatment. Twenty BM samples from patients of benign disorders such as hypersplenism and idiopathic thrombocytopenic purpura were used as controls. Patients and controls were recruited from Ain Shams University Hospitals (Cairo, Egypt). Informed consent was obtained from all patients, controls, and guardians of children before enrollment. All patients signed an informed consent according to the protocols approved by the local ethics committee. The demographic, clinical, and laboratory data of patients are shown in [Table 1]
|Table 1 Descriptive data of all studied patients and control, comparison between acute myeloid leukemia, acute lymphoblastic leukemia patients, and the controls as regards their CLEC12A expression|
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| Methods|| |
The studied patients were subjected to detailed history taking, thorough physical examination, and diagnostic workup of acute leukemia, including complete blood count on Coulter LH 750 cell counter (Coulter Electronics Ltd, Hialeah, Florida, USA). BM aspiration was performed with examination of Leishman-stained peripheral blood. Flow cytometric immunophenotyping was performed on BM samples using a standard panel of monoclonal antibodies (MoAb) on Coulter Epics XL 3-color flow cytometer (Coulter Electronics Ltd), as well as cytogenetic analysis by fluorescence in-situ hybridization (FISH) and conventional karyotyping (results were obtained from patient reports).
The diagnosis of acute leukemia was based on clinical, morphologic criteria, cytochemical stains, immunophenotyping, and cytogenetics according to the WHO classification .
Peripheral blood and BM aspiration samples were collected under complete aseptic conditions on EDTA, potassium salt (K2-EDTA) (1.2 mg/ml) for complete blood count, and immunophenotyping. For cytogenetic analysis, 2 ml of BM aspirate was collected in tubes coated with lithium heparin.
The EDTA anticoagulated BM samples were kept at ambient temperatures and processed within 24 h of collection. Fifty microliters of each sample was incubated with 5 μl of antihuman CLEC12A-phycoerythrin (PE)-conjugated MoAb supplied by R&D system Inc., Minneapolis, Minnesota, USA, or its isotypic control, for 15 min at room temperature in the dark. Next, the cells were washed with 2 ml of PBS, followed by red cell lysis using 1.5 ml of lysing solution (NH4Cl buffered with KHCO3 at pH 7.2) for 3 min at room temperature in the dark. In AML patients, gating on blast cells was done using CD45-PC5 (supplied by Coulter Electronics Ltd) based on their low expression of CD45 and low SSC properties (CD45 low/SSC low). In the control tube, CD45-PC5/CD34-FITC/CLEC12A-PE MoAbs were added. After appropriate gating on blast cells, surface expression and the median fluorescence intensity (MFI) of CLEC12A were determined. Data acquisition and analysis were performed on Epics XL flow cytometer (Coulter Corporation, Hialeah, Florida, USA) using SYSTEM II, version 3 software (Beckman Coulter, Brea, California, USA) with a standard three-color filter configuration.
Cytogenetic analysis was performed by FISH technique using fluorophore-labeled locus-specific identifier (LSI) dual-color probes (Vysis; Abbott Molecular Diagnostics, Abbott Park, Illinois, USA): LSI PML/RARA dual-color translocation probe to detect t(15;17); LSI AML/ETO dual-color, double-fusion probe to detect t(8;21); LSI CBFβ (16q22) break-apart rearrangement probe to detect inv(16)/t(16;16); LSI MLL dual-color, break-apart probe for 11q23 rearrangement; and LSI dual-color single-fusion BCR/ABL to detect t(9;22) (9q34;22q11). Slides were prepared from material fixed in methanol–acetic acid. All probes were set up separately on different slides for each patient. Hybridization and detection of hybridization signals were performed according to the manufacturer’s protocols. For each probe, at least 100 interphase cells were evaluated using the CytoVision 7.3.1 (Leica Biosystems, Richmond, Virginia, USA) to detect the target abnormalities. Images of FISH were captured through the program Mac Probe 4.4 of Power Gene System (Applied Imaging Corporation, San Jose, CA, USA).
According to the national comprehensive cancer network guidelines , the studied AML patients were classified into three risk groups − favorable risk: t(15;17), inv(16)/t(16;16), or t(8;21); intermediate risk: normal cytogenetics, +8 alone, t(9;11), other nondefined; and poor risk: complex (≥3 clonal chromosomal abnormalities), monosomal karyotype: −5, 5q−, −7, 7q−, t(9;22), t(6,9), inv(3)/t(3;3), 11q23 non-t(9;11).
Data were analyzed using statistical program for social science (SPSS Inc., Chicago, Illinois, USA), version 18 IBM-compatible PC. Quantitative data were described in the form of number and percentage, range, and mean±SD. Qualitative data were described as frequency and percentage. Student’s t-test, Mann–Whitney U-test, and χ2-test were used for intergroup comparison. Pearson’s correlation was used to assess the association between two normally distributed variables, and Spearman’s rank correlation coefficient was used for correlating between data when one or more are skewed. ROC curve was used to find out the overall diagnostic value of CLEC12A and to determine the best cutoff value with detection of sensitivity and specificity at this cutoff value. A P value less than 0.05 was considered significant.
| Results|| |
CLEC12A expression in patients and control
CLEC12A expression was found to be positive in all studied AML patients (100%), including both the newly diagnosed untreated and the relapsed cases [mean percentage expression 74±19.8 (range: 38.5–97.5%), with MFI of 3.71±1.96 (range: 1.11–6.69)]. However, CLEC12A expression was found to be negative in all studied ALL (100%) patients [mean percentage expression of 9.85±3.38 (range: 4–15.1%), with MFI of 1.07±0.14 (range: 0.88–1.38)]. As for control samples, CLEC12A was negatively expressed on CD34+ cells with a mean percentage expression of 7.8±4.1% and an MFI of 1±0.4 ([Figure 1] and [Table 1]).
|Figure 1 Representative figure of CLEC12A expression showing positive expression in a case of acute myeloid leukemia (AML) and negative expression in a case of acute lymphoblastic leukemia (ALL) and normal CD34+ progenitor cells of a control.|
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Both mean CLEC12A percentage expression and MFI were significantly higher in AML patients compared with ALL and controls (P<0.001). In contrast, no significant difference was elicited between ALL and the controls (P=0.356) ([Figure 1] and [Table 1]).
As shown in [Table 2], no significant difference was found between the 44 newly diagnosed untreated AML patients and the 16 AML patients in relapse as regards their mean CLEC12A percentage expression or MFI (P=0.0525 and 0.114, respectively) ([Table 1]).
|Table 2 Relation between CLEC12A expression and the studied demographic, clinical, and laboratory data of acute myeloid leukemia patients|
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ROC curve analysis revealed that CLEC12A % expression at a cutoff value of at least 15.1% can diagnose and differentiate between AML and ALL with 100% sensitivity, 100% specificity, and 100% positive and negative predictive values (area under the curve 100%) ([Figure 2]).
|Figure 2 Receiver operating characteristic curve analysis of CLEC12A expression in acute myeloid leukemia (AML) as a diagnostic marker to differentiate between AML and acute lymphoblastic leukemia.|
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Relation between Clec12A and CD34 expression
In the present study, CD34 positivity was detected in 36 (60%) AML patients, whereas 24 (40%) patients were CD34 negative. Mean CLEC12A % expression on CD34+ AML blasts was 75.1±20.8 (range: 54.1–99.5%), mean MFI was 3.54±1.98 (range: 1.11–6.19) compared with 74.1±21.7 (range: 38.9–94.7%), and MFI was 3.94±2.1 (range: 1.42–6.69) on CD34-blasts. No significant difference was found between CD34-positive and CD34-negative patients as regards their mean CLEC12A % expression or MFI (P=0.899 and 0.609, respectively) ([Table 2]).
Comparison between AML patients as regards CLEC12A expression according to their demographic and laboratory data
As shown in [Table 2], both mean CLEC12A % expression and MFI were significantly higher in children compared with adults, as well as in patients with hepatosplenomegaly (P<0.05). However, no significant difference was detected as regards sex, lymphadenopathy, newly diagnosed untreated patients versus patients in relapse, or different cytogenetic prognostic groups (P>0.05) ([Table 1]).
CLEC12A expression was significantly different between studied FAB subtypes; the highest mean % expression was detected in M4 (88.6±8.5), followed by M1 (85.6±9.3), and the lowest was in M0 (39.5±0.85) (P<0.001). Concerning MFI, the highest mean was detected in M5 (5.34±1.21) patients, whereas the lowest level was detected in M3 (1.26±0.2) patients, with a statistical significant difference between different FAB subtypes (P<0.001) ([Table 2]).
Correlation between CLEC12A and laboratory data of studied AML patients
No significant correlation was found between mean CLEC12A % expression or MFI and WBCs, hemoglobin level, platelets, peripheral blood and BM blasts %, or lactate dehydrogenase (P>0.05).
CLEC12A expression in paired diagnosis/relapse samples
The 44 newly diagnosed untreated AML patients were treated with curative intent and were followed up for 12 months, revealing the incidence of relapse in seven (15.9%) patients including four children and three adult patients. No significant difference was found between mean CLEC12A % expression and MFI at diagnosis and in relapse after treatment (94.6±3.0 vs. 91.2±3.42 for Clec12A %, P=0.132; and 5.65±0.3 vs. 4.9±0.5 for MFI, P=0.596), indicating marker stability at relapse after chemotherapy.
| Discussion|| |
This study highlighted the importance of CLEC12A as a new specific diagnostic marker for identification of acute myeloid leukemia by flow cytometry. Our results revealed that CLEC12A was positively expressed on myeloid leukemia cells of all studied AML (100%) patients, while lacking on ALL leukemia cells. According to ROC curve analysis, CLEC12A at a cutoff of at least 15.1% can diagnose AML with high sensitivity and specificity (100%), strongly suggesting that it can be used to diagnose and differentiate AML from ALL in a routine flow cytometry.
Bakker et al.  observed CLL-1 (CLEC12A) positivity in 92% of their analyzed de novo AML samples, clearly indicating the potential for CLL-1 as a novel target for AML. Larsen et al.  reported hMICL (CLEC12A) restriction to the CD45 low/SSC low population of AML cells (being positive in 89% in the retrospectively collected AML test set, and 94% in the prospectively collected test set with a mean of 92% hMICL-positive cells), but it was absent on lymphoid blasts in all cases studied, strongly suggesting that CLEC12A can be used to diagnose AML from ALL in a routine flow cytometry setting.
To assess the overall applicability of CLEC12A as a flow cytometer tool in AML immunophenotyping, we studied CLEC12A expression in diagnostic BM samples from childhood AML patients and found all samples (100%) to be CLEC12A positive, with a significantly higher percentage expression and MFI compared with adult AML patients. This finding highlights the validity of CLEC12A as a pan AML marker. In agreement with our results, Larsen et al.  studied hMICL expression in six consecutive series of BM samples from childhood AML patients, which were all positive.
To underscore the specificity and applicability of CLEC12A as LAIPs, % expression and MFI were measured in normal CD34+ hematopoietic stem cells from the controls showing lack of CLEC12A expression on normal hematopoietic stem cells, with a mean % expression of 7.4% and MFI ranging from 0.2 to 1.4, which were significantly lower than AML cases. Our findings were in agreement with those of Larsen et al. , who found hMICL to be absent or very infrequently expressed on a subset of CD34+ cells constituting a mean of 0.49% in normal BM, with a significantly higher expression level in AML blasts compared with normal CD34+ hMICL+cells. Bakker et al.  reported that only a minority of CD34+ marrow cells displayed reactivity with CLL-1 antibodies. This finding positions the antigen as a potential therapeutic target that allows eradication of antigen-bearing leukemic cells and the subsequent re-establishment of normal hematopoiesis through the remaining normal stem cells . In addition, van Rhenen et al.  found CLL-1 to be present on AML and absent on normal CD34+/CD38− cells at different time points of disease/treatment, which holds potential to serve as a tool to detect residual leukemic CD34+CD38− cells after chemotherapy and as a possible target for therapy.
AML is generally regarded a stem-cell disease, as CD34 is considered the best surrogate marker for hematopoietic stem cells, and is a widely applied backbone in LAIP identification because of its expression on immature cells, even though CD34 negativity has been reported as high as 20–30% of AML cases at diagnosis when using a cutoff of 1% ,,,. Therefore, CD34− patients can be otherwise poorly lineage-characterized immunophenotypically and difficult to monitor for residual disease. The results of the present study detected CD34 negativity in 40% of studied AML patients; the expression of CLEC12A was measured in both CD34+ and CD34− leukemic blasts, showing no significant difference regarding percentage expression or MFI, paving the way to improve LAIP characterization and minimal residual disease quantification by FCM in both CD34+ and the poorly lineage-characterized CD34− AML patients.
Stability of CLEC12A as a biomarker in AML is an essential parameter; to assess this, we compared paired diagnosis and relapse samples from AML patients who were treated with curative intent, revealing positivity in both samples without loss of CLEC12A expression in any of the relapsed samples, with no significant difference in expression and fluorescence intensity indicating that CLEC12A is a stable LAIP marker during the course of disease and confirming that antigenic shift is, at most, a minor problem with this antigen. This is in contrast to other surface antigens routinely used for AML immunophenotyping, because of their infrequent expression at diagnosis or being prone to change at relapse in 35% of cases ,,,.
| Conclusion|| |
CLEC12A is a reliable, specific, and stable diagnostic marker of both childhood and adult acute myeloid leukemia that could pave the way to improve LAIPs both in CD34+ and the poorly characterized CD34− patients by flow cytometry. In addition, low expression of CLEC12A on normal CD34+ progenitor cells positions the marker as a potential therapeutic target.
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Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2]