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 Table of Contents  
ORIGINAL ARTICLE
Year : 2014  |  Volume : 39  |  Issue : 4  |  Page : 232-237

Quantitative assessment of MLAA-34 expression in a sample of Egyptian patients with acute myeloblastic leukemia


1 Department of Clinical Pathology, Faculty of Medicine, Alexandria University, Alexandria, Egypt
2 Department of Hematology, Faculty of Medicine, Alexandria University, Alexandria, Egypt

Date of Submission28-Jun-2014
Date of Acceptance21-Nov-2014
Date of Web Publication25-Mar-2015

Correspondence Address:
Mona Wagdy Ayad
9 Abd El Hamid El Deeb Street, Sarwat, Alex
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1110-1067.153966

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  Abstract 

Background: MLAA-34 is a novel acute monocytic leukemia-associated antigen. Previous studies found that it might be an antiapoptotic factor and play a role in the carcinogenesis and the progression of leukemia, and its overexpression might be associated with an unfavorable presentation of acute myeloid leukemia (AML). Aim and objectives: The aim of this work was to study the expression of MLAA-34 in AML and its correlation with known prognostic parameters and the clinical outcome. Patients and methods: In 30 patients with de-novo AML compared with a control group of 25 age-matched and sex-matched individuals, MLAA-34 expression was assessed using quantitative real-time RT-PCR. Results and conclusion: Results revealed an overexpression of MLAA-34 in patients with AML-M5 compared with controls. Also, MLAA-34 expression was associated with a poor outcome and unfavorable presentation (a high leukocytic count and extramedullary disease). In conclusion, MLAA-34 may be used as a prognostic tool in AML, especially M5. Egyptian J Haematol 39:-0 © 2014 The Egyptian Society of Haematology.

Keywords: Acute myeloid leukemia, gene expression, MLAA-34 gene


How to cite this article:
El-Bordiny M, El-Ghandour A, Ayad MW, Abou-Diba N. Quantitative assessment of MLAA-34 expression in a sample of Egyptian patients with acute myeloblastic leukemia. Egypt J Haematol 2014;39:232-7

How to cite this URL:
El-Bordiny M, El-Ghandour A, Ayad MW, Abou-Diba N. Quantitative assessment of MLAA-34 expression in a sample of Egyptian patients with acute myeloblastic leukemia. Egypt J Haematol [serial online] 2014 [cited 2019 Nov 13];39:232-7. Available from: http://www.ehj.eg.net/text.asp?2014/39/4/232/153966


  Introduction Top


Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy characterized by clonal expansion of immature hematopoietic cells that accumulate in the bone marrow and peripheral blood, interfering with normal hematopoiesis [1]. AML is the most common leukemia in adults: 60-75% of patients with AML younger than 60 years achieve complete remission after intensive induction therapy with or without bone marrow transplantation; however, more than 50% of these patients will relapse, leading to 5-year survival rates of ~30-40%. Known that the median age of AML patients is 60 years, where results are unfavorable and rate of remission is markedly reduced [2],[3],[4],[5]. There was a strong need for the development of novel adjuvant treatment options to be used in combination with the standard therapy to eliminate residual leukemic cells to eliminate residual leukemic cells, prolong remission and survival rates.

Immunotherapeutic approaches targeting leukemia-associated antigens for the eradication of residual AML cells by the immune system after standard therapy may be a promising novel adjuvant treatment to improve the AML outcome and reduce the risk of relapse [5].

A total of 35 novel antigens were identified on acute monocytic leukemia [6]. MLAA-34 is one of the novel monocytic leukemia-associated antigens (MLAAs) identified, reacting exclusively with sera from allogenic leukemia patients but not with normal donor sera [7].

Analysis of the full cDNA sequence showed that the MLAA-34 gene (GenBank no: Ay288977.2), located on chromosome 13q 14.2, is highly homologous to the known human gene calcium-binding protein like 39 (CAB39L) [8]. The MLAA-34 gene has been confirmed to be a novel splice variant of CAB39L associated with acute monocytic leukemia [9].

With RNA interference technology, down-regulation of MLAA-34 expression suppresses the proliferation of U937 cells in vitro and increases the spontaneous apoptosis of these leukemia cells, indicating that MLAA-34 might be a novel antiapoptotic factor related closely to the carcinogenesis or the progression of AML [8].

The aim of this study was to detect MLAA-34 expression in de-novo AML and to evaluate the relation between the expression levels of MLAA-34 and the known prognostic parameters and clinical outcomes.


  Materials and methods Top


Patients and sample collection

Peripheral blood and bone marrow aspirates of 30 patients with newly diagnosed de-novo AML admitted to the Hematology Department of the Alexandria Main University Hospital, during the period between January 2012 and February 2013, were obtained before induction chemotherapy, after patients gave informed consent according to the Alexandria Faculty of Medicine code of ethics. Patients with AML M3 (acute promyelocytic leukemia) were excluded from the study.

The diagnosis of AML and its subtypes was determined according to the French-American-British (FAB) classification.

Peripheral blood and bone marrow samples were obtained from 25 participants of matching age and sex with no history of any malignancies or chemotherapy or irradiation exposures as a control group.

All AML patients received induction chemotherapy using the 3+7 protocol [10]. Follow-up by day 28 bone marrow samples was performed to detect the response to treatment.

Five AML patients were excluded from the study because we could not follow them.

Response definition

Complete remission was defined as bone marrow blasts of 5% or less, an absence of extramedullary disease, absolute neutrophil count more than 1000 cell/ml and platelet count more than 100 000 cell/ml, and independence of red cell transfusion [11],[12].

RNA isolation, cDNA synthesis, and RT-PCR

Purification of the total cellular RNA from human whole blood was performed using the QIAamp RNA blood Mini kit (Qiagen, Hilden, Germany). QIAamp spin columns represent a technology for total RNA preparation that combines the selective binding properties of a silica-based membrane with the speed and the convenience of microspin technology. A specialized high-salt buffering system allows RNA species longer than 200 bases to bind to the QIAamp membrane.

The cDNA was synthesized using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, Massachusetts, USA). In brief, 10 ml of RNA was reverse transcribed into cDNA in a 20 ml reaction using a random hexamer for 10 min at 25°C, 120 min at 37°C, and 5 min at 85°C using a Touchgene gradient thermal cycler, (TECHNE Cambridge, United Kingdom).

Real-time quantitative PCR assay

TaqMan-based real-time PCR (RT-PCR) technology was used [13]. Relative quantitation of the MLAA-34 gene expression was normalized to the endogenous gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH); it was performed by RT-PCR, using the real-time cycler Rotor gene Q (Qiagen) and the ready-to-use QuantiFast probe two-step RT-PCR assay (Qiagen). The PCR mixture contained 12.5 ml of QuantiFast multiplex PCR master mix (HotStar Taq plus DNA polymerase, QuantiFast Probe PCR Buffer and dNTP mix), 1.25 ml of QuantiFast primers and probe assay for the target gene, 1.25 ml QuantiFast primers and probe assay for the house-keeping gene, RNase-free water, and cDNA (≤100 ml/reaction) in a total reaction volume of 25 ml. PCR was performed under the following conditions: 95°C for 5 min followed by 45 cycles of 95°C for 30 s and 60°C for 30 s. A negative control was included in each experiment. Data were presented as 2 -∆∆Ct , where Ct was the threshold cycle and ∆Ct was the Ct value of the target amplification minus that of the reference amplification [13],{14}.

Statistical analysis

Statistical analysis was carried out using SPSS statistics software (IBM Corp. Released 2011. IBM SPSS Statistics for Windows, Version 20.0. Armonk, NY), version 20. Categorical variables were described using frequencies and percentages. The c2 -test was used for testing associations between categorical variables. Quantitative data were tested for normality using the Kolmogorov-Smirnov test. Normally distributed variables were described using mean and SD, and in such cases, the independent sample t-test was used for comparing two groups. Abnormally distributed data were given as the median (minimum-maximum). Nonparametric statistical tests of significance were applied; the Mann-Whitney test was used to compare two independent groups and the Kruskal-Wallis test was used to compare three independent groups. Any significant Kruskal-Wallis comparison was followed by adjusted post-hoc pair-wise comparisons. Pearson's correlation was used for testing correlations between quantitative variables. Statistical significance was accepted as P-vaue less than 0.05. All applied statistical tests of significance were two tailed.

Receiver operating characteristic (ROC), carried out using MedCalc Statistical Software, was used to evaluate the diagnostic and prognostic accuracy of a test to pick diseased and nondiseased individuals correctly. The Youden index was used to find the cutoff point that yields the maximum correct classification.


  Results Top


MLAA-34 gene expression in cases compared with controls

MLAA-34 gene expression was compared between AML patients and controls using 2 -∆∆Ct . AML patients showed significant overexpression of MLAA-34 compared with controls, with a mean expression level of 44.17 ± 42.69 (range 1.04-161.38) for patients and 1.10 ± 0.60 (range 0.19-2.59) for controls [Figure 1].
Figure 1: A box plot showing the expression of MLAA-34 in patients and controls. The upper and the lower borders of the box represent the 25th and the 75th percentiles, and the horizontal line inside the box represents the median (50th percentile). Circles represent outliers. MLAA, monocytic leukemia-associated antigen.

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Receiver operating characteristic (ROC) curve analysis for MLAA-34 expression

ROC levels in AML patients and normal controls revealed that MLAA-34 expression can significantly discriminate cases from controls. It has an excellent diagnostic performance [area under the curve (AUC) = 0.975, 95% confidence interval (CI) = 0.891, 0.998, P < 0.0001]. By the Youden index (J), the cutoff point was 1.6, with a sensitivity of 96.67% (95% CI = 82.8, 99.9) and a specificity of 92% (95% CI = 74, 99). The positive predicative value (PPV) was 93.5% (95% CI of PPV = 78.28, 99.2) and the negative predicative value (NPV) was 88.5% (95% CI of NPV = 69.4, 97.7) [Figure 2].
Figure 2: ROC curve analysis using MLAA-34 expression levels for discriminating AML patients from normal controls. MLAA, monocytic leukemia-associated antigen; ROC, receiver operating characteristic.

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MLAA-34 expression in AML cases in relation to the clinical outcome

The clinical outcome was significantly associated with the MLAA-34 expression level (H = 13.181, P < 0.001). Post-hoc paired comparisons revealed that the median expression level of MLAA-34 of patients in remission was significantly lower than that of patients who failed to achieve complete remission (adjusted P < 0.05); however, the MLAA-34 expression level of patients who died was not significantly different from that of patients who achieved remission or who did not (adjusted P>0.05) [Table 1] and [Figure 3].
Figure 3: The relation between relative quantitative expression and the clinical outcome.

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Table 1: MLAA-34 expression in AML patients in relation to the clinical outcome

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The ROC curve analysis was used to evaluate the sensitivity and the specificity of MLAA-34 in the detection of remission among AML cases

Analysis revealed that the relative quantitative expression of MLAA-34 was excellent in detecting remission (AUC = 0.909, 95% CI = 0.720, 0.987, P < 0.0001). Using the Youden index, J = 14.76 is the cutoff point below which remission was detected with a sensitivity of 75% (95% CI = 42.8, 94.5) and a specificity of 100% (95% CI = 73.5, 100). The PPV was 100% (95% CI of PPV = 66.4, 100) and the NPV was 80% (95% CI of NPV = 51.9, 95.7) [Figure 4].
Figure 4: ROC curve analysis for the clinical outcome among AML patients. AML, acute myeloid leukemia; ROC, receiver operating characteristic.

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The relation between MLAA-34 expression and FAB subtypes

Regarding FAB subtypes, it was found that MLAA-34 expression was higher in patients with AML-M5 compared with the other AML subtypes (M1, M2, and M4) (P = 0.025) [Figure 5].
Figure 5: The relation between the MLAA-34 expression level and the FAB subtype. FAB, French-American-British; MLAA, monocytic leukemia-associated antigen.

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The relation of MLAA-34 expression and extramedullary disease at presentation

MLAA-34 showed significant overexpression among AML cases with extramedullary presentation at diagnosis (P = 0.006) [Table 2].
Table 2: The relation between MLAA-34 expression and extramedullary presentations

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Correlation between the MLAA-34 expression level and the age

There was no significant correlation between MLAA-34 expression and the age (r = -0.83, P = 0.547).

Relation between the MLAA-34 expression level and the sex

There was no significant relation between MLAA-34 expression and the sex (P = 0.228).

Correlation between the MLAA-34 expression level and hematological parameters

A statistically significant positive correlation was evident between MLAA-34 expression and the white blood cell count (r = 0.444, P = 0.001). However, there was a statistically significant negative correlation between MLAA-34 expression and the platelet count (r = −0.389, P = 0.003). MLAA-34 showed no significant correlation with the hemoglobin level (r = −0.258, P = 0.062) or the bone marrow blast percentage (r = −0.093, P = 0.003) [Figure 6].
Figure 6: A scatter plot for the relationship between MLAA-34 expression and (a) the WBC count and (b) platelets. MLAA, monocytic leukemia-associated antigen; WBC, white blood cell.

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  Discussion Top


During the last decade, several leukemia-associated antigens were identified that induce specific T-cell responses in AML. MLAA-34 is a novel antigen; there are few publications regarding the pattern of MLAA-34 expression in AML and its correlation with known prognostic parameters. To our knowledge, this is the first study among Egyptian patients.

The aim of the current study was to investigate MLAA-34 expression levels at diagnosis of de-novo AML, especially acute monocytic leukemia (AML-M5), using RT quantitative PCR and to correlate between its expression levels and clinical outcomes after induction chemotherapy.

In agreement with Zhao et al. [5], it was found that the relative quantitative expression of MLAA-34 was significantly lower in the control samples in comparison with AML cases (P < 0.001). ROC analysis for the diagnostic power of MLAA-34 was performed and it showed that MLAA-34 could differentiate AML cases from controls with 96.67% sensitivity and 92% specificity at a cutoff value of 1.6. The AUC value was 0.975 (P < 0.001), which indicates a good discriminating power.

The discrepancy in the expression levels of MLAA-34 between AML patients and normal controls agrees with Zhang et al. [8], who stated that MLAA-34 might be an antiapoptotic factor closely related to the carcinogenesis or the progression of AML. The resistance of tumor cells to apoptosis may be associated with unfavorable features at presentation and a poorer response to treatment. Hence, MLAA-34 can be considered as an appropriate target for immunotherapy in AML patients, which attacks leukemic cells without affecting normal cells.

Regarding FAB AML subtypes, MLAA-34 expression was significantly higher among the AML-M5 subtype in comparison with other AML subtypes (P = 0.025). This may denote that MLAA-34 is mainly a MLAA.

MLAA-34 over-expression was associated with unfavorable clinical features at presentation (a high leukocytic count and extramedullary presentation: P = 0.001 and 0.006, respectively). This is supported by Zhao et al. [5], and it suggests that MLAA-34 overexpression is accompanied by the high proliferative nature of leukemic blasts.

Considering the response to induction chemotherapy, it is known that early assessment of the sensitivity to chemotherapy represents a tool to predict prognosis. In the current study, evaluation of the MLAA-34 expression level in relation to the day 28 response to chemotherapy revealed a statistically significant relation (P < 0.001) between MLAA-34 overexpression and an unfavorable response to induction chemotherapy.

ROC analysis was used to evaluate the sensitivity and the specificity of MLAA-34 in the detection of remission among AML cases. It showed that MLAA-34 could detect remission with 75% sensitivity and 100% specificity at cutoff value of 14.76. The AUC value was 0.909 (P < 0.001), which indicates a good discriminating power.

This suggests that MLAA-34 might be an antiapoptotic factor and its overexpression is associated with the high proliferative nature of leukemic blasts, delayed blast clearance from the bone marrow or the peripheral blood, and the resistance of blasts to apoptotic stimuli provided by chemotherapeutic agents. This will eventually lead to a poor overall survival and relapse-free survival, which need thorough validation by further studies.

MLAA-34 expression may be used as a prognostic marker for AML, especially AML-M5. Further studies should be carried out including a larger group of patients and refractory and relapsed AML. Follow-up should be for a longer duration, taking into consideration the overall survival, the correlation between MLAA-34 expression and FLT3, and cytogenetic analysis.


  Acknowledgements Top


Conflicts of interest

There are no conflicts of interest.

 
  References Top

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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
 
 
    Tables

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