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 Table of Contents  
ORIGINAL ARTICLE
Year : 2014  |  Volume : 39  |  Issue : 1  |  Page : 32-35

The value of anti-Pax-5 immunostaining in pediatric acute leukemia


1 Departement of Clinical and Chemical Pathology, Faculty of Medicine, Ain Shams University, Cairo, Egypt
2 Department of Clinical and Chemical Pathology, National Research Center, Cairo, Egypt

Date of Submission30-Oct-2013
Date of Acceptance21-Nov-2013
Date of Web Publication29-Jan-2014

Correspondence Address:
Yasmin N ElSakhawy
Department of Clinical and Chemical Pathology, Faculty of Medicine, Ain Shams University, Cairo, 11566
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1110-1067.124844

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  Abstract 

Background Pax-5 protein is a transcription factor expressed in the B-lymphoid lineage from the earliest detectable precursor to the mature B-cell stage. Its expression is closely correlated with B-cell precursor acute lymphoblastic leukemia (pre-B-ALL), which is one of the most common childhood malignancies.
Aim of the work To evaluate the expression of anti-Pax-5 monoclonal antibody on leukemic blasts in pediatric patients with acute leukemia and to compare it with anti-CD20 to show its diagnostic utility.
Patients and methods This study was carried out on 35 pediatric patients diagnosed with acute leukemia, who were divided into two groups: Pre-B-ALL and Acute, myeloid leukemia (AML). All patients were subjected to a complete assessment of history, full clinical and laboratory examination, bone marrow aspirate and biopsy, and immunophenotyping. Immunostaining of bone marrow trephine biopsy specimens using Pax-5 and CD20 monoclonals was carried out for all cases.
Results Pax-5 was positive in 79.1% of cases in the pre-B-ALL group and negative in all AML patients. CD20 was positive in 37.5 and 18% of cases in the ALL and the AML groups, respectively. Pax-5 appeared to be more sensitive (79.2%) and more specific (100%) than CD20 in differentiating ALL and AML.
Conclusion Our results highlight the beneficial role of including Pax-5 in an immunohistochemical panel to diagnose acute leukemia of B-cell lineage.

Keywords: acute leukemia, IHC, Pax-5, pediatrics


How to cite this article:
Shams El Din El Telbany MA, ElSakhawy YN, Sallam MT. The value of anti-Pax-5 immunostaining in pediatric acute leukemia. Egypt J Haematol 2014;39:32-5

How to cite this URL:
Shams El Din El Telbany MA, ElSakhawy YN, Sallam MT. The value of anti-Pax-5 immunostaining in pediatric acute leukemia. Egypt J Haematol [serial online] 2014 [cited 2019 Dec 13];39:32-5. Available from: http://www.ehj.eg.net/text.asp?2014/39/1/32/124844


  Introduction Top


The Pax-5 gene is a member of the paired-box Pax gene family and encodes the B-cell-specific transcription factor, Pax-5 [1] . Within the hematopoietic system, Pax-5 is expressed in the B-lymphoid lineage from the earliest detectable precursor to the mature B-cell stage, but not in plasma cells [2],[3] . Immunostaining for the B-cell-specific transcription factor Pax-5 has been advocated as the most sensitive, specific, and reliable immunohistochemical (IHC) marker for B-cell lineage, and may be particularly useful when CD20 is negative [4],[5] . It is suggested that the inclusion of pax5 in a panel of antibodies to assess undifferentiated malignant neoplasms may have a diagnostic benefit [6] .

Pax-5 deregulation is associated with certain types of acute leukemia, particularly B-cell precursor acute lymphoblastic leukemia (pre-B-ALL), which is one of the most common childhood malignancies [7] . Pax-5 has been shown to be correlated closely with this form of leukemia; its expression in B-ALL closely parallels that of CD79a, the most useful marker currently available for B-ALL diagnosis in routine bone marrow biopsies [6] , and may become a reliable marker for the diagnosis of this disease [8] .

The aim of the present study is to evaluate the expression of anti-Pax-5 monoclonal antibody on leukemic blasts in pediatric patients with acute leukemia and to compare it with anti-CD20 to show its diagnostic utility.


  Materials and methods Top


The present study included 35 pediatric patients diagnosed with acute leukemia referred from hematology and oncology clinics of Ain Shams University Hospitals to hematology laboratory during the period from January 2008 till June 2010. There were 19 males and 16 females, with a male to female ratio of 1.2 : 1.0; their ages ranged from 3 to 16 years. The 35 patients were divided into two groups: Pre-B-ALL and AML. The diagnosis of patients in this study was made on the basis of clinical presentation, laboratory data, bone marrow aspirate, flow cytometry, and cytogenetic study. All patients were subjected to bone marrow trephine biopsies after obtaining written consent from their parents. A core biopsy 1.5-2 cm in length was collected under complete aseptic conditions using sterile trephine biopsy needles. Fixation was performed for 24 h; then, decalcification of the core was performed using disodium EDTA for 48 h. This was followed by passing the core in serial concentrations of ethyl alcohol (50, 70, 85, 90, and 100%), ending with xylene and then wax, followed by paraffin embedding. Serial 3-μm sections were cut from the paraffin block, mounted on positively charged slides, and dried overnight in a 60°C oven. Deparaffinization was performed in xylene for 24 h, followed by hydration in descending grades of alcohol: 100, 90, 85, and 70%. Antigen unmasking was performed by the heat-induced epitope retrieval method using an antigen retrieval solution (Dako, Carpinteria, CA, USA) with pH 6.0 for a period of 20 min in a microwave at 800 W. Endogenous peroxidase activity was blocked by incubation of the tissue section with 3% hydrogen peroxide in water for 30 min, followed by incubation with the primary monoclonal antibody [Table 1]. The streptavidin-biotin method with the horseradish peroxidase enzyme was used as a detection kit (LSAB2; Dako). The sections were then counterstained in Mayer's hematoxylin, coverslipped by DPX mount media, and examined under a light microscope. The positivity of the immunostaining was detected by the percentage of positive cells, where more than 10% was considered positive [Figure 1], [Figure 2], [Figure 3], [Figure 4] [9] .
Table 1: Monoclonal antibodies

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Figure 1: Pre-B ALL case CD20 negative.

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Figure 2: Pre-B ALL case CD20 positive.

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Figure 3: AML case Pax-5 negative.

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Figure 4: Pre-B ALL case Pax-5 positive.

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Statistical analysis

Data were analyzed statistically using the statistical package for social science (version 15.0.1). The following tests were used: Descriptive statistics including quantitative data (mean ± SD) for parametric results and in the form of median and range for nonparametric results and qualitative data (number and percentage), and analytical statistics including χ2 -test and Fisher's exact test. The sensitivity and specificity of the markers were calculated. The sensitivity of a marker was calculated by dividing true positives by both true positives and false negatives and then multiplied by 100, whereas specificity was calculated by dividing true negatives by both true negatives and false positives and then multiplied by 100.


  Results Top


Morphologically, all cases showed diffuse infiltration by blast cells in the bone marrow biopsy. The immunohistochemistry results [Table 2] indicated that Pax-5 was positive in 19/24 (79.1%) of pre-B-ALL cases; however, it was negative in all AML cases. As for CD20, it was positive in 9/24 (37.5%) of ALL cases and in 2/11 (18.0%) of AML cases. Of the 19-positive Pax-5, only nine patients were CD20 positive in the pre-B-ALL group. There was a statistically significant difference between CD20 positivity and pax5 positivity among ALL patients (P < 0.05). There was a significantly higher rate of Pax-5 positivity in ALL cases when compared with AML cases (P = 0.001); however, this statistical significance was absent for CD20 expression in both groups (P = 0.43). Pax-5 appeared to be more sensitive and more specific than CD20 in differentiating ALL and AML [Table 3].
Table 2: Results of Pax-5 and CD20 in both groups

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Table 3: Sensitivity and specificity for both markers in the acute lymphoblastic leukemia group

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  Discussion Top


The diagnosis of acute leukemia depends mainly on bone marrow aspirate samples; however, in some instances, especially with heavy infiltration and dry tap samples, bone marrow aspiration becomes difficult. In these cases, bone marrow biopsy samples and IHC markers are very importance. Of these markers, Pax-5 has been advocated as the most sensitive, specific, and reliable IHC marker for B-cell lineage. Its expression is detectable as early as the pro-B-cell stage and subsequently in all further stages of B-cell development until the plasma cell stage, where it is downregulated. The pax5 gene plays a central role in B-cell development, activation, and differentiation [10] .

In the B-ALL group, 79.1% of patients were positive for the pax5 marker, with 11/24 (45.8%) showing strong intensity of staining and 8/24 (33.3%) showing moderate intensity of staining. In agreement with our results, Zhang et al. [11] found that the Pax5 marker was expressed in 72.4% of the B-ALL patients studied. In addition, Torlakovic et al. [12] , Desouki et al. [13] , and Nasr et al. [14] found that the Pax5 marker was expressed in all the B-ALL patients studied (20 cases, five cases and 34 cases, respectively) with a strong intensity of staining [12-14] . Dong et al. [5] also found that out of 31 cases of pre-B-ALL well-characterized by standard flow cytometry and morphology, Pax-5 was uniformly detected in all tumor cells of all cases including all decalcified bone marrow trephine biopsy specimens.

On comparing pax5 with CD20, the present study results showed that pax5 expression was higher than CD20 in terms of positivity and intensity of staining (79.1 and 37.5%, respectively). Also, Pax-5 appeared to be more sensitive and more specific than CD20 in the ALL group [Table 3].

In agreement with the results of the present study, Torlakovic et al. [12] found that pax5 is an excellent pan B-cell and pan pre-B-cell marker, which showed a sensitivity of 100% when compared with the CD20 marker for benign mature B-cells and their various neoplastic disorders as well as for pre-B-ALL . In addition, Chu et al. [9] carried out a study on CD20-negative B-ALL patients for expression of pax5 as a novel B-lineage-specific marker and found that all the CD20-negative B-ALL cases studied expressed pax5 with a strong intensity of staining. Their results suggest that the expression of Pax-5 precedes the expression of CD20 and that the application of Pax-5 IHC makes it a useful B-lineage marker for B-ALL [9] .

In the AML group, Pax-5 was negative in all the cases studied in the present work. According to Tiacci et al. [15] , Feldman and Dogan [16] , and Dong et al. [5] , Pax-5 was significant in the AML group only if associated with t(8;21) . Valbuena et al. [17] indicated that silencing of Pax-5 is not required for commitment to myeloid differentiation and its expression in AML was found mainly in cases with t(8;21). On revising the cytogenetic study for AML cases in the present study, none of them was found to have t(8;21). For CD20, only 18.1% positivity was detected in the AML group. This was consistent with Chu et al. [9] , who detected 18% positivity, whereas Smith et al. [18] detected a very poor significance in the myeloid lineage with positivity 0.8%.


  Conclusion Top


The Pax-5 marker is a good pre-B-cell marker. It is suggested that the IHC application of this marker is useful as a B-lineage marker for B-ALL. Our results highlight the beneficial role of including Pax-5 in an INH panel to diagnose acute leukemia of the B-cell lineage. However, more studies are needed to detect the expression of Pax-5 in various types of AML.


  Acknowledgements Top


 
  References Top

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2.Barberis A, Widenhorn K, Vitelli L, Busslinger M. A novel B-cell lineage-specific transcription factor present at early but not late stages of differentiation. Genes Dev 1990; 4 :849-859.  Back to cited text no. 2
    
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
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