ORIGINAL ARTICLE Year : 2021  |  Volume : 46  |  Issue : 2  |  Page : 75-82

Diagnostic and prognostic value of neuron-glial antigen 2 expression in adult acute myeloid leukemia

Amr M Gawaly MD 1, Rasha A Elkholy2, Rasha Y Hagag1, Amal E Abd El-Lateef2, Alzahraa A Allam1
1 Department of Internal Medicine, Faculty of Medicine, Tanta University, Tanta, Egypt
2 Department of Clinical Pathology, Faculty of Medicine, Tanta University, Tanta, Egypt

Date of Submission25-Dec-2020Date of Acceptance21-Jan-2020Date of Web Publication29-Oct-2021

Correspondence Address:
Amr M Gawaly
Department of Internal Medicine, Faculty of Medicine, El Gish Street, Tanta, 9111
Egypt

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/ejh.ejh_61_20

Background Acute myeloid leukemia (AML) is a hematopoietic stem cell disorder characterized by a block in differentiation of hematopoiesis, resulting in growth of a clonal population of neoplastic cells or blasts. Neuron-glial antigen 2 (NG2) is not expressed by normal hematopoietic stem cells but expressed on blast cells in adult AML. NG2 has been incorporated in diagnostic panels for immunophenotyping of leukemic patients because of its positive predictive value for Mixed Lineage Leukemia (MLL) rearrangements.
Aims To assess NG2 expression in adult patients with AML and its correlation with disease-free survival.
Patients and methods A total of 60 patients were divided into two groups: 40 newly diagnosed patients with AML and 20 patients diagnosed as having hypersplenism used as a control group. Leukemic patients were diagnosed on the basis of clinical presentation, morphological and cytochemical examination of peripheral blood and bone marrow smears, as well as immunophenotyping criteria for diagnosis of AML. NG2 expression was evaluated in the two groups using flow cytometry.
Results No significant differences were found in both age and sex in different patient groups. NG2 expression in the patient group versus control group showed a statistically significant difference. There was no statistically significant difference regarding complete blood count, lactate dehydrogenase, and erythrocyte sedimentation rate, as well as blast percentage in the peripheral blood and in the bone marrow on comparing NG2-positive group and NG2-negative group. There is a significant increase in disease-free survival and overall survival in the negative NG2 expression than in the positive NG2 expression group (P=0.043).
Conclusion NG2 expression has a major role in the outcome of patients with AML. NG2 expression analysis can be used as a prognostic marker in newly diagnosed patients with AML. NG2 could be a target for therapy by using anti-NG2 antibody in a subset of patients with AML who do not respond to conventional therapy.

Keywords: acute myeloid leukemia, MLL rearrangements, neuron-glial antigen 2

How to cite this article:
Gawaly AM, Elkholy RA, Hagag RY, Abd El-Lateef AE, Allam AA. Diagnostic and prognostic value of neuron-glial antigen 2 expression in adult acute myeloid leukemia. Egypt J Haematol 2021;46:75-82
How to cite this URL:
Gawaly AM, Elkholy RA, Hagag RY, Abd El-Lateef AE, Allam AA. Diagnostic and prognostic value of neuron-glial antigen 2 expression in adult acute myeloid leukemia. Egypt J Haematol [serial online] 2021 [cited 2021 Oct 30];46:75-82. Available from: http://www.ehj.eg.net/text.asp?2021/46/2/75/329512   Introduction 

Acute myeloid leukemia (AML) represents a group of biologically heterogeneous clonal proliferation of undifferentiated myeloid precursor cells with diverse clinical and biological features [1].

Chromosomal aberrations are known to be of high prognostic significance. Moreover, flow cytometric and molecular genetics parameters have contributed to a new classification of AML [2].

Despite the advances in therapeutic approaches such as intensive-chemotherapy as well as stem cell transplantation, which have dramatically improved the treatment outcome for some patients, the overall survival (OS) rate is still low, and only 40–50% of patients with AML can be cured, and many do not achieve long-term remission [3].

There are many prognostic factors that can affect AML response and disease-free survival (DFS). The most important are the treatment regimen, age, white blood cell count, immunophenotyping, cytogenetic, and molecular genetics determined at the time of diagnosis. Continuous research studies aim to improve treatment outcome by optimizing dosage and scheduling of anti-leukemic agents based on individual patient’s leukemic cell phenotypic and genetic features, in addition to host normal cell pharmacogenetic characteristics [4].

Neuron-glial antigen 2 (NG2) also known as melanoma chondroitin sulfate proteoglycan [5], is a transmembrane proteoglycan presents on the surface of more than 90% of malignant melanomas in addition to some nonmelanomic tumors. Moreover, NG2 is expressed on glioma cells, as well as on developing and adult oligodendrocyte precursor cells [6].

The NG2 oligodendrocyte progenitors are activated by demyelination rather than by inflammation [7]. This fact may be related to the frequent Central Nervous System (CNS) involvement in Mixed Lineage Leukemia (MLL) rearrangement.

NG2 is not expressed by normal hematopoietic cells but is selectively expressed by leukemic blast cells [8]. NG2 has been incorporated in the diagnostic panels of acute leukemia by immunophenotyping because of its positive predictive value for MLL rearrangements in AML and acute lymphoblastic leukemia (ALL) [9].

These AML blasts show predominately French American-British (FAB) M5 morphology and have 11q23/MLL gene rearrangement aberrations [8].

In addition, NG2 expression has been evaluated concerning the detection of persisting minimal residual disease [9].

Expression of NG2 can be used as a prognostic marker for acral lentiginous melanoma and infantile AML [10].

It was already shown that NG2 mAb does not react with normal hematopoietic cells but only with leukemic cells of AML or ALL with 11q23/MLL gene rearrangement aberrations, in an attempt to further characterize AML cells and to contribute prognostically new data [2].

The aim of this study was to evaluate the expression of NG2 on adult AML cells and assess its diagnostic and prognostic effect in these patients.

  Patients and methods 

The current study was carried out from July 2018 to December 2019 on 40 patients with newly diagnosed AML admitted to Hematology/Oncology Unit, Internal Medicine Department, Tanta University Hospitals, Egypt. They were 22 (55%) males and 18 (45%) females, and their ages ranged from 15 to 75 years. A total of 20 patients who presented with pancytopenia and were diagnosed as having hypersplenism were used as a control group. There were 10 (50%) males and 10 (50%) females, and their ages ranged from 18 to 65 years. This study was carried out according to the Declaration of Helsinki. After research ethical committee approval, an informed written consent was taken from all patients.

Patients were subjected to the following: full history taking, careful clinical examination, abdominal ultrasonography, and laboratory investigations, including the following:

Routine investigations:Complete blood count with examination of Giemsa-stained peripheral smears.Bone marrow (BM) aspiration with examination of Giemsa-stained smears and cytochemical-stained smears (MPO and Sudan black).Immunophenotyping for AML diagnosis was performed using the following monoclonal antibodies:Myeloid lineage markers: CD 13 PE, CD 33 PE, CD 117 PE, cyt anti-MPO PE, glycophorin A PE, CD 71 FITC, and CD61 FITC.Monocytic lineage markers: CD 14 PE and CD 64 FITC.B-lymphoid lineage markers: CD 10 PE, CD19 PE, anti-TDT FITC, and cyt CD 79a PE.T-lymphoid lineage markers: CD 2 FITC, CD7 FITC, and cyt CD3 APC.Nonspecific lineage markers: CD 45 Per CP, HLA-DR FITC, and CD 34 FITC.These markers were supplied by Becton Dickinson (Mountain View, California, USA).Erythrocyte sedimentation rate.Lactate dehydrogenase level determination.Specific investigation:

For identification of cells expressing NG2, flow cytometric analysis of BM samples of all patients was done using PE-labeled anti-human NG2 monoclonal antibody, BD Bioscience Company (BD Biosciences, 2350 Qume Drive San Jose, CA, USA), CN: 562415, and also PE mouse IgG2a isotype was used to prevent nonspecific binding.

Cutoff for neuron-glial antigen 2 expression was more than or equal to 5%

Samples were analyzed using Becton Dickinson (BD) FACS Caliber instrument (Becton Dickinson, San Diego, California, USA), and Cell Quest software (Becton Dickinson, version 3, verify software House Topsham, Maine, USA).

Follow-up of the patients

After being fully investigated, all the patients received chemotherapy and were observed for 6 months regarding clinical and laboratory findings of remission and relapse, taking care to estimate the date of first complete remission, date of relapse, and date of death or last seen alive.

Statistical analysis

The collected data were organized, tabulated, and statistically analyzed using SPSS software (Statistical Package for the Social Sciences, version 13; SPSS Inc., Chicago, Illinois, USA). Test of univariate normality was done using Kolmogorov–Smirnov test. For qualitative data, comparison between two groups was done using χ2 test and Fisher exact test. Independent t test was used to compare two groups for quantitative normally distributed variables. Mann–Whitney test was used to compare two groups for abnormally distributed quantitative variables. Receiver operating characteristic (ROC) curve was used to determine the diagnostic performance of the markers. Area more than 50% gives acceptable performance, and area about 100% is the best performance for the test. Kaplan–Meier survival curve was used, and Cox regression was done for the significant relation with DFS and OS. Significance of the obtained results was judged at the P value less than or equal to 0.05 level [11].

  Results 

This study was conducted on 40 newly diagnosed patients with AML. There were 22 (55%) males and 18 (45%) females, with male to female ratio of 1.2 : 1. Moreover, 20 patients who presented with pancytopenia and diagnosed as having hypersplenism were involved in the study as a control group to evaluate the normal range of NG2 expression. There were 10 (50%) male and 10 (50%) females, with a male to female ratio of 1 : 1. There were insignificant difference between both groups regarding sex.

Age of the patient group ranged from 15 to 75 years, with a mean value of 14.725±17.235 and that of the control group ranged from 18 to 65 years, with a mean value of 39.700±16.288. There was no statistically significant difference between both groups regarding age.

Regarding clinical signs observed in the AML patient group, the results were as follows: hepatosplenomegaly was observed in 18 (45%) patients, fever was observed in 18 (45%) patients, lymphadenopathy was observed in six (15%) patients, and bleeding was observed in 10 (25%) patients. Clinical data of patient and control groups are shown in [Table 1].

Regarding FAB classification observed in AML group, the results were as follows: M0 was observed in two (5%) patients, M1 in six (15%) patients, M2 in six (15%) patients, M3 in four (10%) patients, M4 in six (15%) patients, M5 in 12 (30%) patients, and M7 in four (10%) patients.

Laboratory data of patients and control groups are shown in [Table 2].

Table 2 Comparison between patient group and control group regarding laboratory data

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ROC curve analysis was done to detect cutoff for NG2 expression and showed that NG2 expression more than or equal to 5% had a sensitivity of 75%, a specificity of 100%, a positive predictive value of 100%, and a negative predictive value of 66.67%, and the area under the curve was 0.882, as shown in [Table 3] and [Figure 1].

Table 3 Agreement (sensitivity and specificity) for neuron-glial antigen 2 expression to predict cases (vs. control)

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Figure 1 ROC curve for NG2 expression to predict cases (versus control). NG2, neuron-glial antigen 2; ROC, receiver operating characteristic.

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NG2 expression by flow cytometry was as follows: in the patient group, 10 (25%) patients showed negative NG2 expression (<5%), and 30 (75%) patients showed positive NG2 expression (≥5%) ([Figure 2] and [Figure 3]), whereas all patients in the control group showed negative NG2 expression. The difference between both groups was statistically significant, as shown in [Table 4] and [Table 5].

Figure 2 Dot blot shows positive NG2 expression. NG2, neuron-glial antigen 2.

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Figure 3 Dot blot shows negative NG2 expression. NG2, neuron-glial antigen 2.

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Table 4 Neuron-glial antigen 2 distribution of expression in patient group versus control group

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Table 5 Neuron-glial antigen 2 range of expression in patient group versus control group

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There was no statistically significant difference between positive NG2 group and negative NG2 group regarding complete blood count data, lactate dehydrogenase, and erythrocyte sedimentation rate, as well as blast percentage in both peripheral blood and BM, as shown in [Table 6].

Table 6 Comparison between neuron-glial antigen 2 positive group and neuron-glial antigen 2 negative group regarding laboratory data

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Moreover, on comparison between NG2 positive group and NG2 negative group regarding FAB classification, there were no statistically significant differences, as shown in [Table 7].

Table 7 Comparison between neuron-glial antigen 2 positive group and neuron-glial antigen 2 negative group regarding French American-British classification

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The 40 newly diagnosed patients with AML were treated and followed up for 6 months. On follow-up of the studied patients, we observed that 10 patients died (two with NG2 negative expression and eight with NG2 positive expression), 12 patients showed complete remission (eight with NG2 negative expression and four with NG2 positive expression), and 18 patients showed relapse (all of them were positive for NG2 expression). There is significant increased DFS and OS in the negative NG2 group than in the positive NG2 group using Kaplan–Meier survival curve. Distribution of the studied cases regarding survival is provided in [Table 8],[Table 9],[Table 10] and [Figure 4] and [Figure 5].

Table 8 Comparison between neuron-glial antigen 2 positive group and neuron-glial antigen 2 negative group regarding the outcome of acute myeloid leukemia group

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Figure 4 Kaplen–Meier survival curve showed significant increased disease-free survival in the negative NG2 than in the positive NG2 expression group. NG2, neuron-glial antigen 2.

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Figure 5 Kaplen–Meier survival curve showed significant increased overall survival in the negative NG2 than in the positive NG2 expression group. NG2, neuron-glial antigen 2.

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  Discussion 

AML comprises a heterogeneous group of clonal stem cell diseases with variable clinical outcome [2]. The discovery of new disease modifier or prognostic factor can help to tailor AML treatment and improve patient survival.

Expression of NG2 is not restricted to melanoma but also expressed on solid tumors such as tumors of neuroectodermal origin, squamous cell carcinoma of the head and neck, basal breast cancer, mesothelioma, pancreatic carcinoma, some types of renal cell carcinoma, chordoma, chondrosarcoma, and soft tissue sarcomas [12].

NG2 is not expressed by normal hematopoietic stem cells; however, it was found to be expressed on blast cells of adult AML. NG2 has been incorporated in the diagnostic panel of acute leukemia because it was found in ∼90% of acute leukemia with MLL rearrangement [13].

Bueno et al. [14] found that NG2 antigen is expressed in CD 34+HPCs and plasma-cytodendritic cell precursors only if leukemogenesis is triggered regardless the type of leukemia.

NG2 is type I a single-pass transmembrane protein. It has three domains: a large extracellular, transmembrane, and intracellular domain [15]. The extracellular one interacts with many extracellular stromal proteins such as laminins, collagen, P-selectin, and integrin. This gives NG2 molecule an important role in adhesion of the blast cells to the BM stroma [16]. Blockade of the cellular interaction of NG2 helps blast cell mobilization from BM to the peripheral blood. Interaction of leukemic cells with the BM microenvironment is one of the most important mechanism in leukemic cell survival, growth, relapse, and chemoresistance [13].

The expression of NG2 in ALL is extensively studied; however, its expression in AML is still needed for more studies to document or rule out its significance in AML diagnosis and prognosis.

A total of 40 patients with newly diagnosed AML and 20 control patients with pancytopenia diagnosed as having hypersplenism were included in our study. They presented to the Internal Medicine Department in Tanta University Hospital. The ages of the patient group ranged from 15 to 75 years and control group ranged from 18 to 65 years.

Based on ROC curve analysis, NG2 expression more than or equal to 5% is used as a cutoff for positivity for this marker, with sensitivity 75%, specificity 100%, and area under the curve 0.882, which denote high diagnostic efficacy. This agrees with Petrovici and colleagues who reported more than or equal to 5%of the gated cells as a cutoff value for NG2 positivity on leukemic cells.

All patients in control group showed negative NG2 expression by flow cytometry, whereas in the patient group, most patients (75%) showed positive NG2 expression (≥5%). The difference between both groups was statistically significant. So, NG2 expression can be used as a marker for discrimination between normal and malignant myeloblast.

In this study, NG2 positivity was higher in AML/M5 (33.33%), followed by M2 (20%), and the least positive cases were associated with M1 (6.67%). M3, M4, and M7 showed 13.33% positivity rate. Our finding agrees with Petrovici and colleagues who reported that NG2 is highly expressed in patients with AML M5 subtype, but they reported absent expression in immature AML subtypes.

A worse prognosis in patients with AML having positive NG2 expression than those with negative NG2 expression was observed in this study. NG2-positive patients showed shorter DFS and OS than patients with negative NG2 expression. These results agreed with Petrovici and colleagues, who reported that NG2 is highly expressed in patients with AML with bad prognosis, whereas in patients with good prognosis, NG2 expression was lower. These data raise the possibility that anti-NG2-targeted therapy could be a potential option for this group of patients. Anti-NG2 therapeutic agents mobilize the blast cells from the BM to the peripheral blood. This makes the blast cells more accessible and more sensitive to chemotherapy.

Acknowledgements

Xth Eurasian hematology oncology conference, Istanbul, Turkey, October 8–11, 2019.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8], [Table 9], [Table 10]