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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 44  |  Issue : 4  |  Page : 237-245

Positive expression of FOXP1 protein as a good prognostic factor in childhood acute lymphoblastic leukemia: a retrospective study


1 Department of Clinical Pathology, Faculty of Medicine, Asyut University, Egypt
2 Department of Clinical Pathology, South Cancer Institute, Assiut University, Egypt

Date of Submission03-Sep-2019
Date of Acceptance17-Nov-2019
Date of Web Publication20-Jul-2020

Correspondence Address:
Marwa Saad Mohamed
Department of Clinical Pathology, Faculty of Medicine, Assiut University
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ejh.ejh_37_19

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  Abstract 


Background Deficiency of FOXP1 in early B-lymphoid precursors results in a block at the transition of pro-B to pre-B-cell stage and severely reduces the peripheral mature B-cell compartment.
Objective The objective of the current study was to detect the prognostic value of positive FOXP1 protein expression on acute lymphoblastic leukemia (ALL) in children and adults evaluated by disease-free survival (DFS) and overall survival and its relation with BCR-ABL fusion gene in those patient.
Materials and methods A retrospective study was carried out on 50 bone marrow samples from fixed bone marrow aspirates taken from patients previously diagnosed with ALL at South Egypt Cancer Institute. They were classified into 25 BCR-ABL-positive samples and 25 BCR-ABL-negative samples.
Results Positive expression of FOXP1 protein in B lymphocytes in patients with ALL was associated with better overall survival and DFS, especially in children. Coexistence of both FOXP1 and BCR-ABL proteins in the patients with ALL was associated with good prognosis than BCR-ABL-positive patients with negative expression of FOXP1.
Conclusion The positive expression of FOXP1 protein in B lymphocytes in pediatric patients with ALL and its association with better overall survival and DFS indicate that FOXP1 protein expression in pediatric ALL is a good prognostic marker. Coexistence of both FOXP1 and BCR-ABL proteins in the patients with ALL appeared as a good prognostic factor.

Keywords: acute lymphoblastic leukemia, BCR-ABL, FOXP1 protein, tyrosine kinase inhibitors


How to cite this article:
Helal SR, Mosad E, Abd-Elrahman MZ, ElGammal S, Mohamed MS. Positive expression of FOXP1 protein as a good prognostic factor in childhood acute lymphoblastic leukemia: a retrospective study. Egypt J Haematol 2019;44:237-45

How to cite this URL:
Helal SR, Mosad E, Abd-Elrahman MZ, ElGammal S, Mohamed MS. Positive expression of FOXP1 protein as a good prognostic factor in childhood acute lymphoblastic leukemia: a retrospective study. Egypt J Haematol [serial online] 2019 [cited 2020 Aug 8];44:237-45. Available from: http://www.ehj.eg.net/text.asp?2019/44/4/237/290233




  Introduction Top


Acute lymphoblastic leukemia (ALL) is a clonal proliferation of lymphoid progenitor cells in the bone marrow, blood, and extramedullary sites. Although ALL occurs 80% in children, it has a bad prognosis when it occurs in adults [1].

FOXP1 is widely expressed in several tissues with highest levels in lymphocytes, neural tissue, and gut. It is a critical transcriptional regulator during B-lymphopoiesis [2],[3]

In 2018, the study by Thomas Patzelt et al. [4] confirmed the role of FOXP1 in B-cell development, as it was found that deficiency of FOXP1 in early lymphoid precursors results in reduction in the peripheral mature B-cell compartment, as it cause a block at the transition of pro-B to pre-B cell stage.

The t(9;22)/Philadelphia chromosome (Ph+) is present in only 3–5% of children with ALL [5]. In adult, ALL can range from 15 to 50% and increases with age. Ph positivity has implications in terms of both prognosis and treatment. Historically, Ph-positive ALL has a 1-year survival of around 10%. However, with the development of tyrosine kinase inhibitors (TKIs), survival has improved, and thus the Ph status of all patients must be obtained before starting therapy [1].

It was found that Gpr132 gene is a target for FOXP1 action, and overexpression of FOXP1 in a pre-B cell line resulted in increased transcription of Gpr132 gene, which encodes the G-protein-coupled receptor G2A protein, which acts as a tumor suppressor in mouse pre-B cells, where it antagonizes the effect of BCR-ABL [6].

BCR‐ABL1‐like B‐lymphoblastic leukemia (BCR‐ABL1‐like B‐ALL) is a neoplasm of B lymphoblasts that demonstrates a characteristic pattern of gene expression similar to that of BCR‐ABL1-positive B‐ALL but lacks the BCR‐ABL1 translocation. FOXP1 was found as one of the fusion partners of ABL1 [7]. It is a tyrosine kinase fusion predictive for activated ABL signaling [8].


  Materials and methods Top


This study is a retrospective study in which 50 bone marrow samples from fixed bone marrow aspirates taken from patients previously diagnosed with ALL at South Egypt Cancer Institute were enrolled. Informed consent was taken from all patients. It was explained to all patients the study objectives and that the collected data (names, nature of disease and any information about the patients) will be confidential and for the purpose of scientific research only. The research was not invasive, not harmful and without any complication. BCR-ABL fusion gene was already previously determined for these aspirates, and they were classified according to it into 25 BCR-ABL-positive samples and 25 BCR-ABL-negative samples. Patients were diagnosed with ALL according to the presence of greater than 20% blasts in the peripheral blood and/or bone marrow [8], morphologic assessment of Leishman-stained bone marrow aspirate smears, hematoxylin and eosin-stained bone marrow core biopsy, flow cytometry with immunophenotyping for CD19, CD22, CD79a, Tdt, and CD10 positive in common ALL, cytoplasmic Ig positive in pre-B ALL, surface Ig positive in mature B ALL, and cytogenetic for BCR-ABL fusion gene by fluorescent in-situ hybridization technique.

Detection of FOXP1 protein was done by indirect immunofluorescence technique using a fluorescence microscope with FITC filters and DAPI filters.

Indirect immunofluorescence technique for detection of FOXP1 protein

Overall, 50 µl of fixed bone marrow cells was dropped on to a slide. Specimens were incubated for 30 min with 10% normal blocking serum. Then the slides were washed with PBS three times for 5 min each. Then the slides were incubated with 50 µl of primary antibody for 60 min at room temperature using antibody concentration 1 : 10 diluted in PBS. The slides were washed with PBS three times for 5 min each. The slides were incubated again at room temperature for 60 min, in a dark chamber, with 50 µl of a fluorophore-conjugated secondary antibody. The slides were washed with PBS three times for 5 min each. The slides were stained by DAPI as a counterstain. The cover slip was mounted with mounting medium. The slide was examined by using a fluorescence microscope with FTIC filters and DAPI filters (type: Carl Zeiss AxioSkop 2 Mot FL, Carl Zeiss Oy Niittyvillankuja 4B 01510 Vantaa, Finland; Camera type: Leica CW 4000 fluorescent in-situ hybridization version 1.1 (Leica Biosystems Division of Leica Microsystems Inc., 1700 Leider Lane Buffalo Grove, IL, United States), and Software: CarlZeiss/Cytoversion, Axioversion control 3.1). We used 30% as a cutoff to define positivity of B lymphocytes for FOXP1 protein expression ([Figure 1]).
Figure 1 (a) Indirect immunofluorescence of a bone marrow sample of one of the studied patients with acute lymphoblastic leukemia showed negative FOXP1 protein expression; (b) indirect immunofluorescence of a bone marrow sample of one of the patients with acute lymphoblastic leukemia showed positive FOXP1 protein expression.

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Statistical analysis

Statistical testing with χ2 analysis was used to examine relationships between variables. Overall survival was the period from the date of diagnosis until death from any cause or the last follow-up, and the disease-free survival (DFS) was the period during and after treatment in which a patient is living with a disease that does not get worse. Survival curves were examined by the Kaplan–Meier method, using log rank to analyze the differences between groups.


  Results Top


There was a higher percentage of ALL in males (56%) compared with females (44%). Moreover, there was a higher percentage of ALL in children (64%) over adults (36%).

In the studied patients with ALL, there were 25 (50%) patients who were BCR-ABL positive and 25 (50%) patients who were BCR-ABL negative.

In this study, 20 of 50 patients with ALL (40%) showed negative expression of FOXP1 protein in their bone marrow aspirate in contrast to 30 of 50 patients with ALL (60%) who showed positive expression.

In this study, the percentage of FOXP1 protein expression in all patients ranged from 3–90%, with a mean of 30.43. Percentage of FOXP1 protein-positive expression ranged from 32–91%, with a mean of 61%. Percentage of positive FOXP1 in positive BCR-ABL patients ranged from 32 to 91%, with a mean 60%, whereas the percentage of positive FOXP1 in negative BCR-ABL patients ranged from 32 to 87%, with a mean of 65% ([Table 1]).
Table 1 Percentage of FOXP1 protein expression in the studied patients with acute lymphoblastic leukemia

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There was a significant positive correlation between BCR-ABL gene and FOXP1 protein expression in the patients, as BCR-ABL-positive patients were associated with positive expression of FOXP1 in 68% out of them compared with 32% with negative expression of FOXP1 (P=0.049) ([Table 2]).
Table 2 The correlation between BCR-ABL fusion gene and FOXP1 protein expression

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In this study, BCR-ABL-positive patients showed improvement in overall survival and DFS compared with BCR-ABL-negative patients ([Figure 2]).
Figure 2 (a) Kaplan–Meier curve comparing the overall survival of positive and negative BCR-ABL fusion gene in the studied patients with acute lymphoblastic leukemia; (b) Kaplan–Meier curve comparing the disease-free survival of positive and negative BCR-ABL fusion gene in the studied patients with acute lymphoblastic leukemia.

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Pediatric BCR-ABL-positive patients treated by tyrosine kinase inhibitors showed a significant improvement in overall survival and DFS compared with BCR-ABL-negative patients who showed inferior overall survival and DFS ([Figure 3]).
Figure 3 (a) Kaplan–Meier curves comparing the overall survival of positive and negative BCR-ABL fusion gene in the studied children with acute lymphoblastic leukemia; (b) Kaplan–Meier curve comparing the disease-free survival of positive and negative BCR-ABL fusion gene in the studied children with acute lymphoblastic leukemia; (c) Kaplan–Meier curve comparing the overall survival of positive and negative BCR-ABL fusion gene in the studied adult patients with acute lymphoblastic leukemia; (d) Kaplan–Meier curve comparing the disease-free survival of positive and negative BCR-ABL fusion gene in the studied adult patients with acute lymphoblastic leukemia.

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Adult BCR-ABL-positive patients treated with tyrosine kinase inhibitors showed a significant improvement in DFS compared with BCR-ABL-negative patients who showed inferior DFS, but there was no significant difference in overall survival between the two groups ([Figure 3]).

FOXP1 positivity appeared as a good prognostic factor as these patients showed improvement in overall survival and DFS than FOXP1-negative patients ([Figure 4]).
Figure 4 (a) Kaplan–Meier curve comparing the overall survival of positive and negative FOXP1 protein expression in the studied patients with acute lymphoblastic leukemia; (b) Kaplan–Meier curve comparing the disease-free survival of positive and negative FOXP1 protein expression in the studied patients with acute lymphoblastic leukemia.

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Children with ALL who exhibited positive expression of FOXP1 showed a significant improvement in overall survival and DFS in comparison with children with ALL who showed negative expression of FOXP1 ([Figure 5]).
Figure 5 (a) Kaplan–Meier curve comparing the overall survival of positive and negative FOXP1 protein expression in the studied children with acute lymphoblastic leukemia; (b) Kaplan–Meier curve comparing the disease-free survival of positive and negative FOXP1 protein expression in the studied children with acute lymphoblastic leukemia; (c) Kaplan–Meier curve comparing the overall survival of positive and negative FOXP1 protein expression in the studied adult patients with acute lymphoblastic leukemia; (d) Kaplan–Meier curve comparing the disease-free survival of positive and negative FOXP1 protein expression in the studied adult patients with acute lymphoblastic leukemia.

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However, in adult patients with ALL, there was no significant difference in the overall survival and DFS between adult patients with ALL with positive and negative expression of FOXP1 ([Figure 5]).

There was an improvement in overall survival and DFS rates in BCR-ABL positive/FOXP1 positive patient group compared with BCR-ABL negative/FOXP1 negative patient group ([Figure 6]).
Figure 6 (a) Kaplan–Meier curve comparing the overall survival of patients group with BCR-ABL positive/FOXP1 positive and those with BCR-ABL positive/FOXP1 negative; (b) Kaplan–Meier curve comparing the disease-free survival of patients group with BCR-ABL positive/FOXP1 positive and those with BCR-ABL positive/FOXP1 negative.

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  Discussion Top


In this study, we hypothesized that expression of FOXP1 in patients with ALL affects the prognosis of the disease.

There was a higher percentage of ALL in males (56%) over females (44%). This is in agreement with the study by Ketan Kulkarni et al. [9], which found that the majority were male (55%) among patients with ALL.

Moreover, the study by Snodgrass et al. [10] found that incidence rates in children aged 0–4 were 7.55 and 3.32 cases per 100 000 person-years for males and females, respectively. The ratio of males to females diagnosed with ALL was 1.59. There was a higher percentage of ALL in children (64%) over adults (36%). This is in agreement with the study by Snodgrass et al. [10], which found that the incidence rates peaked in children aged 0–4 years, and more than 50% of cases occurring in children 10 years of age or younger. Moreover, this agrees with the literature, and in most countries, the incidence rate of ALL in children is approximately three to four times that in adults.

In this study, patients were divided into 25 (50%) patients positive for BCR-ABL fusion gene and 25 (50%) patients negative for BCR-ABL fusion gene.

In this study, BCR-ABL-positive patients were on tyrosine kinase inhibitors. They showed improvement in overall survival, with a mean of 14.89±3.3 months, and DFS, with a mean of 9.22±1.26 months compared with BCR-ABL-negative patients who showed inferior overall survival, with a mean 10.22±2.55 months, and DFS, with a mean of 4.24±1.1 months. Pediatric BCR-ABL-positive patient group on tyrosine kinase inhibitors showed significant improvement in overall survival and DFS compared with BCR-ABL-negative patients who showed inferior overall survival and DFS. However, adult BCR-ABL-positive patient group on tyrosine kinase inhibitors showed significant improvement in DFS compared with BCR-ABL-negative patients who showed inferior DFS, but there was no significant difference in overall survival between the two groups.

Relatively few studies specifically investigated whether TKI can overcome the negative effect of the Philadelphia chromosome on the prognosis of elderly patients with ALL by direct comparison; however, the study by Byun et al. [11] revealed the possible favorable effect of Philadelphia chromosome positivity in elderly patients with ALL in the TKI era. It reported improved survival for Ph+ ALL group compared with Ph-ALL group. During the follow-up of 33 months, the median overall survival was 11.7 months for the Ph+ group, whereas it was 7.7 months for the Ph- group.

The study by Schultz et al. [5] confirms that children and adolescents with Ph+ ALL treated with intensive chemotherapy with addition of intensive imatinib have an excellent outcome, which appears to stabilize to ∼70–75% long-term outcomes for these patients with a 4-year DFS of 75.2% for good-risk patients receiving imatinib compared with 55.9% for those who did not.

In contrast, the study by Judith M. Boer and colleagues worked on a subset of BCR-ABL-negative cases that have a gene expression profile similar to that of BCR-ABL-positive B-ALL described as Ph-like B-ALL and associated with unfavorable prognosis. It found that tyrosine kinase fusions include kinase‐activating gene rearrangements are only found in patients with BCR-ABL1-like features. The DFS of tyrosine kinase-activating fusion cases was unfavorable compared with fusion-negative cases of ALL.

FOXP1 was found as one of fusion partners of ABL1 [7]. It is a tyrosine kinase fusion predictive for activated ABL [8].

In this study, 30 out of 50 patients with ALL (60%) were positive for FOXP1 protein in their bone marrow aspirate in contrast to 20 of 50 patients with ALL (40%) who had negative expression.

The study by Thomas Patzelt et al. [4] found that siRNA or miR-34a overexpression in bone marrow resulted in downregulation of FOXP1, which resulted in a partial developmental B cell block and diminished mature B-cell population.

In this study, FOXP1 protein positivity appeared as a good prognostic factor specifically in childhood ALL, who showed a significant improvement in overall survival and DFS in comparison with children with ALL who showed negative expression of FOXP1. However, in adult patients with ALL, there was no significant difference in the overall survival and DFS between adult patients with ALL with positive and negative expression of FOXP1 protein.

This is more or less in agreement with the study by Klampfl et al. [12] which showed that decreased expression of FOXP1 is also an unfavorable factor in Philadelphia negative myeloproliferative disorders and the study by Xiao et al. [13] showed that the decrease in expression of FOXP1 is an unfavorable factor in breast cancer, endometrial cancer, NSCLC, prostate cancer, colorectal cancer, and epithelial ovarian cancer.

In this study, the patient group with BCR-ABL positivity and FOXP1 positivity showed improvement in overall survival and DFS compared with the patient group with BCR-ABL positivity and FOXP1 negativity.

This can be explained by the study by Bond et al. [6] which showed that Gpr132 gene is a target for activation by FOXP1 through direct binding to the gene which encodes the G2A protein which was originally identified as an orphan G protein-coupled receptor. The overexpression of FOXP1 in pre-B cells results in increased Gpr132 transcription that increases the G2A protein, which acts as a tumor suppressor in mouse pre-B cells, where it antagonizes the effect of BCR-ABL.

It was found that G2A is transcriptionally induced in B lymphoid progenitors by BCR-ABL during this transformation process and suppresses its transformation potency when overexpressed. G2A is described as a powerful negative modifier of BCR-ABL-mediated leukemogenesis. It was found that mice transplanted with BCR-ABL-transduced G2A-deficient bone marrow cells exhibit earlier tumor onset, more rapid tumor progression, and shorter survival times compared with those receiving BCR-ABL-transduced wild-type bone marrow cells [14].

So, this study’s hypothesis that improvement in overall survival and DFS of the patient group with BCR-ABL positivity and FOXP1 positivity may be explained by action of FOXP1 protein on its target Gpr132 gene that encodes the G2A protein that has been shown to act as a tumor suppressor and antagonizes the effect of BCR-ABL. Coexistence of both FOXP1 and BCR-ABL proteins in the patients of ALL is associated with better overall survival and DFS. A large-scale study is recommended in a larger number of cases and for long-term follow-up. Further molecular and genetic studies on FOXP1 in ALL are necessary to identify molecular mechanisms involved in this process.


  Conclusion Top


The positive expression of FOXP1 protein in B lymphocytes in pediatric patients with ALL and its association with better overall survival and DFS indicate that FOXP1 protein expression in pediatric ALL is a good prognostic marker. Coexistence of both FOXP1 and BCR-ABL proteins in the patients of ALL appeared as a good prognostic factor.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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Terwilliger T, Abdul-Hay M. Acute lymphoblastic leukemia: a comprehensive review and2017 update. Blood Cancer J 2017; 7:e577.  Back to cited text no. 1
    
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Shu W, Yang H, Zhang L, Lu MM, Morrisey EE. Characterization of a new subfamily of winged-helix/Fork head (FOX) genes that are expressed in the lung and act as a transcriptional repressors. J Biol Chem 2001; 276:27488–27497.  Back to cited text no. 3
    
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Bond J, Domaschenz R, Roman-Trufero M, Sabbattini P, Ferreiros-Vidal I, Gerrard G et al. Direct interaction of Ikaros and FOXP1 modulates expression of the G protein-coupled receptor G2A in B-lymphocytes and acute lymphoblastic leukemia. Oncotarget 2016; 7:65923–65936.  Back to cited text no. 6
    
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Siegele BJ, Nardi V. Laboratory testing in BCR-ABL1-like (Philadelphia-like) B-lymphoblastic leukemia/lymphoma. Am J Hematol 2018; 93:971–977.  Back to cited text no. 7
    
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Boer JM, Steeghs EM, Marchante JR, Boeree A, Beaudoin JJ, Beverloo HB et al. Tyrosine kinase fusion genes in pediatric BCR-ABL1-like acute lymphoblastic leukemia. Oncotarget 2017; 8:4618–4628.  Back to cited text no. 8
    
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Kulkarni K, Stobart K, Witol A, Rosychuk RJ. Leukemia and lymphoma incidence in children in Alberta, Canada: a population-based 22-year retrospective study. Pediatr Hematol Oncol 2015; 8:649–660.  Back to cited text no. 9
    
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Snodgrass R, Nguyen LT, Guo M et al. Incidence of acute lymphocytic leukemia in Calgary, Alberta, Canada: a retrospective cohort study. BMC Res Notes 2018; 11:104. doi:10.1186/s13104-018-3225-9  Back to cited text no. 10
    
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Byun JM, Koh Y, Shin DY, Kim, Yoon, Lee JO et al. BCR-ABL translocation as a favorable prognostic factor in elderly patients with acute lymphoblastic leukemia in the era of potent tyrosine kinase inhibitors. Haematologica 2017; 102:e187–e190.  Back to cited text no. 11
    
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Klampfl T, Harutyunyan A, Berg T, Gisslinger B, Schalling M, Bagienski K et al. Genome integrity of myeloproliferative neoplasms in chronic phase and during disease progression. Blood 2011; 118:1.  Back to cited text no. 12
    
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Xiao J, He B, Zou Y, Chen X, Lu X, Xie M et al. Prognostic value of decreased FOXP1 protein expression in various tumors: a systematic review and meta-analysis. Sci Rep 2016; 6:30437.  Back to cited text no. 13
    
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Le LQ, Kabarowski JH, Wong S, Nguyen K, Nguyen K, Gambhir SS, Witte ON. Positron emission tomography imaging analysis of G2A as a negative modifier of lymphoid leukemogenesis initiated by the BCR-ABL oncogene. Cancer Cell 2002; 1:381–391.  Back to cited text no. 14
    


    Figures

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