|Year : 2015 | Volume
| Issue : 3 | Page : 121-129
The SALL4 gene in acute leukemias
Hasnaa A Abo-Elwafa MD 1, Shereen P Aziz1, Maged M Salah2, Osamah B Sedek2
1 Department of Clinical Pathology, Sohag Faculty of Medicine, Sohag University, Sohag, Egypt
2 Department of Clinical Pathology, Assuit Faculty of Medicine, Assuit University, Assuit, Egypt
|Date of Submission||02-May-2015|
|Date of Acceptance||17-May-2015|
|Date of Web Publication||8-Sep-2015|
Hasnaa A Abo-Elwafa
Clinical Pathology, Sohag Faculty of Medicine, Sohag University Hospital, Sohag
Source of Support: None, Conflict of Interest: None
Background The SALL gene family is involved in normal development as well as tumor genesis. SALL4 is essential for the maintenance of the pluripotent and the self-renewal properties of embryonic stem cells.
Patients and methods It was conducted from the period of April 2008 to June 2012 on 40 patients with acute leukemia (group I), 20 patients who had nonmalignant hematologic disease (group II), and 20 healthy individuals (group III). They were subjected to laboratory investigations including a complete blood picture, RT-PCR for the detection of the SALL4 gene, for group I bone marrow aspiration biopsy, cytochemical studies, and immunophenotyping of either peripheral blood or bone marrow samples to diagnose and classify the hematologic malignancy.
Results SALL4 mRNA was expressed in 50% of the acute myeloid leukemia (AML) cases and was undetectable in 50% of the cases. Whereas it was expressed in only 20% of acute lymphoblastic leukemia (ALL), it was undetectable in 80% of ALL. SALL4 mRNA was undetectable in all cases of immune thrombocytopenic purpura (group II) (0%). SALL4 mRNA was undetectable in all samples of the control group (group III) (0%). There was no significant difference between the expression level of CD markers and SALL4 in AML cases, whereas there was a significant elevation of the CD10 expression level in SALL4-positive ALL cases (P < 0.05). There was a significant reduction in the RBC count and the hematocrite (Hct) and platelet level in SALL4-positive ALL cases (P < 0.05), and there was a statistically significant reduction in the platelet count in SALL4-positive AML cases.
Conclusion SALL4 mRNA expression was higher in cases of AML (50%), compared with ALL cases (20%). There was no SALL4 mRNA expression detected in all cases of immune thrombocytopenic purpura patients and in normal control individuals.
Keywords: SALL-4 gene, expression, acute, leukemias
|How to cite this article:|
Abo-Elwafa HA, Aziz SP, Salah MM, Sedek OB. The SALL4 gene in acute leukemias. Egypt J Haematol 2015;40:121-9
| Introduction|| |
The SALL gene family, SALL1, SALL2, SALL3, and SALL4, was originally cloned on the basis of its DNA sequence homology to Drosophila spalt (sal)  . In humans, the SALL gene family is involved in normal development as well as tumor genesis  . SALL1 induces angiogenesis by stimulating VEGF-A promoter activity  . SALL4 is essential for the maintenance of the pluripotent and the self-renewal properties of embryonic stem cells (ESCs) by interacting with two other key regulators in ESCs, NANOG, and OCT4  . Cytogenetic location: Long arm of chromosome 20, region 1 band 3.2 (20q13.2). It has been shown that the large repertoire of splice isoforms expressed in ESCs is gradually reduced in absolute number during neural differentiation  . In addition, during both neural and cardiac differentiation, the specific repertoire of splice isoforms present in pluripotent against committed lineages changes  . Zhen et al.  demonstrated that the two isoforms had overlapping binding sites of both SALL4a/SALL4b, and SALL4b alone are enriched for pluripotency genes, whereas SALL4 alone predominantly binds to the differentiation and patterning gene  .
The ESC factor, SALL4, plays an essential role in both development and leukemogenesis  .
SALL4a and SALL4b collaborate in the maintenance of the pluripotent state, but play a distinct role  .
The oncogenic potential of SALL4 has been recently revealed by multiple studies. The SALL4b transgenic mice exhibit myelodysplastic-like features including ineffective hematopoiesis and an increased proportion of immature cells  . The functional categories of SALL4 target genes in leukemic cells revealed that SALL4 frequently binds to target genes involving various apoptotic pathways, including p53, BCL2, TNF, and PTEN  . During normal hematopoiesis, SALL4 isoforms are expressed in the CD34+ hemopoietic stem cell (HSC)/hemopoietic pluripotent stem cell (HPC) population and are rapidly turned off (SALL4b) or downregulated (SALL4a) in the normal human bone marrow and the peripheral blood. In contrast, SALL4 was constitutively expressed in acute myeloid leukemia (AML), and failed to turn off in human primary AML and myeloid leukemia cell lines  . In AML, BMI1 was demonstrated to be a direct target gene of SALL4, which is expressed constitutively in human leukemia cell lines and primary AML cells. BMI1 phosphorylation results in the dissociation of BMI1 from chromatin, followed by derepression of target genes  .
| Participants and methods|| |
This study was approved by the ethical committee of the Sohag University Hospital. It was conducted on 60 patients and 20 healthy persons as a control group during the period from April 2008 to June 2012.
Participants were classified into the following groups:
- Group I: 20 patients with AML (eight female and 12 male), their age ranging from 6 to 60 years, with a mean ± SD of 28.2 ± 18 years, and 20 patients with acute lymphoblastic leukemia (ALL) (10 female and 10 male), their age ranging from 5 to 42 years, with a mean ± SD of 19.9 ± 11.15 years. The patients were selected according to the laboratory diagnosis of leukemia regardless of their age and sex.
- Group II: 20 patients who had nonmalignant hematologic disease [immune thrombocytopenic purpura (ITP)], their age ranging from 4 to 53 years, with a mean ± SD of 17.75 ± 13 years.
- Group III: 20 healthy individuals, their age ranging from 5 to 50 years, with a mean ± SD of 25.2 ± 12.99 years.
A written consent was taken from all patients and control individuals.
All the studied groups were subjected to the following:
- History taking.
- Thorough clinical examination.
- Abdominal ultrasonography and computerized tomography in some cases.
- Laboratory investigations including:
- Routine investigations including a complete blood picture.
- Reverse transcriptase (RT)-PCR for the detection of the SALL4 gene.
For group I, the following were performed: bone marrow aspiration and bone marrow biopsy, cytochemical studies, and immunophenotyping of either the peripheral blood or the bone marrow samples to diagnose and classify the hematologic malignancy.
- Three milliliter of venous blood was drawn from all patient and control groups into a K 3 EDTA tube for complete blood count on Cell Dyne-1700 (Abbott Diagnostics, 5440 Patrick Henry Dr., Santa Clara, California, USA), and routine chemistry was performed after centrifugation on Synchron CX-9 (Houston, Texas, USA).
- Three milliliter of bone marrow aspirate were collected into a K 3 EDTA vacutainer, and used immediately for immunophenotyping and RNA extraction for the gene expression study.
- A few drops on slides to spread peripheral blood films.
Bone marrow examination
About 2-3 ml of marrow aspirate were collected after bone marrow biopsy on K 3 EDTA vacutainers, and used immediately for immunophenotyping and RNA extraction.
Fresh bone marrow films were prepared, slides were stained with Leishman's stain for examination of the morphology, and others were used for cytochemical staining.
Flow cytometric studies (immunophenotyping)
Bone marrow or peripheral blood samples collected from leukemia patients were subjected to monoclonal antibody fluorescence staining, followed by acquisition and analysis by a FACScaliber flow cytometer (Used BD Beckton Dickinson FACS Caliber Flow Cytometer in Oceanside, California, USA). Antibodies specific to lymphoid-associated antigens (CD3, CD7, CD10, CD19, CD22) and myeloid/monocytic/erythroid antigens (CD13, CD14, CD33, CD235a) were included, in addition to non-lineage-specific antigens human leukocytic antigen (HLA)-DR, CD34, and CD45. They were labeled with phycoerythrin (PE) and fluorescein isothiocyanate (FITC) as follows:
- CD45 FITC/CD34 PE combination.
- CD19 FITC/CD10 PE.
- CD3 PE/CD13 FITC.
- CD7 FITC/HLA-DR (PE).
- CD22 PE/CD33 FITC.
- CD14 FITC.
- CD235a PE.
Antibodies were purchased from Becton and Dickinson (San Jose, California, USA), Coulter Diagnostics, Beckman Coulter, Inc. flow, 250 South, Kraemer Boulevard, Brea, CA 2821-6232, USA; Coulter Clone), Dako (Carpentaria, California, USA), and Serotec (Oxford, UK).
Results were considered positive when 20% or more of the malignant cells expressed a particular antigen (10% or more in case of CD10 expression).
Expression of SALL4 gene in hematopoietic tissues by reverse transcriptase-polymerase chain reaction
PCR primers used for the amplification of the SALL4 gene and the β-actin gene were synthesized (Bio Basic Inc., Canada). Primer sets for both SALL4 and β-actin were designed to span exon/intron boundaries to prevent amplification from any possible contaminating DNA. The SALL4 primer set was designed to amplify sequences common to all alternative splice variants of the gene. The primers were designed as follows:
(1) Primer sequences for the SALL4 gene (National Center for Biotechnology Information NCBI Annotation Project, 2002; AY170622):
(a) SALL4F (forward primer):
Sequence: 5′-CATGATGGCTTCCTTAGATGCC CCAG-3′.
(b) SALL4R (reverse primer):
Sequence: 5′-CCGTGTGTCATGTAGTGAACCTT TAAG-3′.
(2) Primer sequences for the β-actin gene  :
(a) β-Actin forward:
(b) β-Actin reverse:
Sequence: 3′-CGTCATACTCCTGCTTGCTGAT CCACATCTGC-5′.
All primers were reconstituted in diethylpyrocarbonate (DEPC) water to a concentration of 100 mmol/l.
About 2-3 ml of bone marrow aspirate was collected into a K 3 EDTA vacutainer from patients of group Ia and group Ib and group II. About 3 ml of peripheral blood was collected into a K 3 EDTA vacutainer from healthy controls in group III.
The samples were treated immediately by ficoll (lymphocyte separation media, Lymphosep, Cat no. L0560; Biowest, Oklahoma City, OK location has 1 key person listed in Mid-Continent Edition , Bio West Remediation, Bob's Parts & Services American Corp).
RNA extraction from hematopoietic cells
The RNA extraction kit (Cat no. BSC52S2; Bioer) provides a very simple, fast, and economical technique to isolate high-quality RNA and can go high throughput.
- Fresh blood or bone marrow was centrifuged at 3000 rpm for 10 min, and soon after it was taken out, the white layer was absorbed using the ficoll as discussed in the sample preparation, 100 μl/2×10 6 cells were obtained, transferred to a new 1.5-ml tube, and 100 μl of solution R1 were added, mixed thoroughly for 30 s, and incubated at room temperature for 1 min.
- The sample was homogenized and 600 μl of solution R2 were added and mixed thoroughly; the mixture was incubated at the room temperature for 3-5 min.
- The supernatant was transferred into a spin column and centrifuged at 12 000-14 000 rpm at room temperature for 30 s.
- The flow-through was discarded. About 600 μl of wash buffer were added into the spin column, and centrifuged for 30 s. This step was repeated once again and centrifuged for 2 min.
- The spin column was transferred to a sterile 1.5-ml microcentrifuge tube. About 20-50 μl of elution buffer (or RNase-free water pH>7.0) were added to the central part of the membrane, incubated at room temperature for 1 min, and centrifuged for 30 s. The buffer in the microcentrifuge tube was containing the total RNA.
- The total RNA was used directly or stored at 4°C or in the ice bath to be used for RT-PCR or be stored at -80°C.
Reverse transcription and polymerase chain reaction
The two-step protocol for reverse transcriptase-polymerase chain reaction
The BioRT cDNA First-Strand Synthesis Kit (Hangzhou Bioer Technology Co. Ltd; Cat no. BSB09M1, Hangzhou Bioer Technology Co., Ltd, BIN AN RD. HI TECH by Panjiva, United States. Country of Origin ,China. Port Name Location, TOLUCA, ESTADO DE MEXICO., Original Consignee City, Benito Juarez) provides the materials needed to synthesize first-strand cDNA from RNA rapidly and reliably. It uses avian myeloblastosis virus reverse transcriptase (AMV) RT to synthesize first-strand cDNA from the RNA population.
The kit includes all the necessary reagents for first-strand cDNA synthesis. The kit includes both random primers and oligo-dT18 primers. In this study, random primers were used. The RT in the BioRT cDNA First-Strand Synthesis Kit is AMV, which provides up to 60°C RT temperature and provides a higher sensitivity and a higher yield to cDNAs synthesis and PCR. The kit can synthesize up to 10-kb cDNA. The reaction buffer was optimized for this kind of reaction system.
Under extreme care and strict sterile conditions, to prevent the introduction of exogenous RNases, one-time gloves were used; masks were used because of RNases in the saliva and the skin, and sterile filter pipette tips were used to avoid carry over contamination of stock solutions. All consumables were treated at 180°C for 60 min or 37°C for 12 h with 0.1% DEPC H 2 O followed by sterilization at 121°C for 30 min.
Reverse transcriptase reaction
- The reaction setup for cDNA synthesis was as follows: in a microcentrifuge tube, 10 μl of mixture was distributed as follows:
- 1 μl of 10× RT buffer.
- 1 μl of dNTP mixture (10 mmol/l).
- 0.5 μl of random hexamer primer.
- 0.5 μl of RNase inhibitor (40 U/μl).
- 0.5 μl of AMV RT (5 U/μl).
- 5 μl of sample RNA.
- 1.5 μl of RNase-free H 2 O.
- RT reactions were kept at room temperature for 10 min, and then they were transferred to a thermal cycler and incubated at 42°C for 45 min. This was followed by incubation at 95°C for 5 min to inactivate and denature AMV RT completely. They were kept in an ice bath for 5 min before the cDNA was ready for PCR reaction.
Polymerase chain reaction
The PCR mixture was prepared in a microcentrifuge tube on ice as follows:
- 5 μl of 10× reaction buffer.
- 1 μl of dNTP mixture (10 mmol/l each).
- 1 μl of sense primer.
- 1 μl of antisense primer.
- 2 μl of 25 mmol/l MgCl 2 .
- 5 μl of sample DNA.
- 0.5 μl of FIREPol DNA polymerase.
- Deionized PCR-grade water was added to adjust the final volume to 50 μl. The tubes were tapped for good mixing of the content and kept on ice till it was transferred to the thermal cycler.
For each patient sample, two tubes were prepared: one for the SALL4 gene and the other for the β-actin gene, which was used as a control reaction for each sample.
The negative control reaction was prepared to test for DNA contamination: it was prepared by preparing the PCR mixture without a DNA template.
The following PCR cycles profile was conducted:
- One cycle of denaturation at 95°C for 4 min.
- Thirty-five cycles of denaturation at 95°C for 30 s.
- Annealing at 57°C for 30 s.
- Extension at 72°C for 1 min.
One cycle for the final extension step at 72°C for 10 min was performed, and the reaction was held at 4°C.
Electrophoresis and visualization of products
The SALL4 gene and β-actin amplicons were analyzed by electrophoresis on 2% agarose gel at 130 V for 30 min, and visualized by ethidium bromide staining and UV transillumination.
- 2% agarose in 0.5% Tris borate EDTA (TBE) buffer.
- TBE 5×: 54 g Tris base, 27.5 g boric acid, and 20 ml 0.5 mol/l EDTA (pH 8).
- Ethidium bromide staining (2 μl of 10 mg/ml).
- DNA molecular size marker (PCR marker; Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711, USA).
| Results|| |
The study was conducted on 40 patients (22 female and 18 male) who suffered from acute leukemia and another 20 patients with nonmalignant hematological disease (ITP). Their age ranged from 4 to 60 years, with a mean ± SD value of 20.9 ± 14.32 years. Twenty matched healthy individuals (12 male and eight female) were included as a reference control (group III). Their age ranged from 5 to 50 years, with a mean ± SD value of 25.2 ± 12.99.
The studied participants were classified as follows:
- Group I: 40 patients who had acute leukemia. Their age ranged from 5 to 60 years, with a mean ± SD value of 24.05 ± 15.19 years.
- Group II: 20 patients who had nonmalignant hematological diseases (ITP). Their age ranged from 4 to 53 years, with a mean ± SD value of 17.75 ± 13 years.
- Group III: 20 apparently healthy individuals; their age ranged from 5 to 50 years, with a mean ± SD value of 25.2 ± 12.99 years. The patient and the control groups were selected according to the laboratory diagnosis regardless of their age and sex.
Clinical data of the control and the patient groups are represented in [Table 1].
White blood cells
The white blood cell count of the control (group III) group ranged from 4.48 to 9.9 × 10 9 /l, with a mean ± SD value of 6.6 ± 1.47 ([Table 2] and [Table 3]).
The mean value of white blood cells revealed a statistically highly significant elevation in group I and a moderately significant elevation in group II when compared with the control group (P < 0.0001 and <0.01), and a highly statistically significant elevation in group I when compared with group II (P < 0.001).
The mean value of hemoglobin revealed a statistically significant reduction in group I and a moderately significant reduction in group II when compared with the control group (P < 0.001 and <0.01) and a statistically highly significant reduction in group I when compared with group II (P < 0.001).
A statistically highly significant reduction in the platelet count was noted in group Ia and group Ib when compared with the control group (P < 0.0001 and <0.001).
The mean value of platelets revealed a statistically highly significant reduction in group I and group II when compared with the control group (P < 0.0001 and <0.001), and a statistically significant reduction in group II when compared with group I (P < 0.001).
According to the french american british (FAB) classification, bone marrow (BM) films stained by Leishman's stain demonstrated the morphology of M2 subtype in AML cases, L1 and L2 morphology in ALL cases, and L3 morphology in no case ([Figure 1]a and [Figure 2]a).
|Figure 1 (a) BM films stained with Leishman's stain demonstrate the morphology of case number 1 of acute lymphoblastic leukemia (ALL)-L1 and case number 6 of ALL. (b) A BM film stained with Leishman's stain demonstrates the morphology of case number 3 of the acute myeloid leukemia (AML)-M2 subtype.|
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|Figure 2 (a) BM films stained with Leishman's stain demonstrate the morphology of case number 6 of acute lymphoblastic leukemia (ALL)-L2 (left) and case number 1 of ALL-L1 (right). (b) B.M. film stained by PAS and demonstrated strong positivity in case no.1 of ALL-L1.|
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Cytochemical reactions performed for the leukemic patients included myeloperoxidase (MPO), Sudan black B (SBB), periodic acid Sciff (PAS), and nonspecific esterase (NSE), and the following results were obtained ([Figure 1]b and [Figure 2]b):
In AML cases, MPO and SBB reaction were positive in 14 (70%) cases and negative in six (30%) cases. The PAS reaction was positive in two (10%) cases and negative in 18 (90%) cases and the NSE reaction was positive in 30% and negative in 70% of the cases.
[Figure 1]b showed a BM film with a strong MPO reaction in the AML-M2 subtype, a strong SBB-positive reaction in the same case, and a BM film with a positive NSE reaction in the AML-M5 subtype.
The MPO reaction was negative in all cases (100%). The SBB reaction was negative in all cases (100%). The PAS reaction was positive (70%) and the NSE reaction was negative in all cases (100%).
[Figure 2]b shows a BM film with a strong PAS reaction in case of the ALL-L2 subtype.
Immunophenotyping in acute leukemia patients (group I)
The expression level of CD45 in AML patients ranged from 29 to 99%, with a mean ± SD of 78.03 ± 24.13 years, whereas the level of CD45 in ALL patients ranged from 12.5 to 99%, with a mean ± SD of 58.64 ± 26.2 years ([Table 4] and [Figure 3]).
|Figure 3 Immunophenotyping in leukemic groups. AML, acute myeloid leukemia; ALL, acute lymphoblastic leukemia.|
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|Table 4 Sall-4 gene in acute lymphoblastic leukemia in comparison to immunophenotyping|
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The expression level of CD34 in AML patients ranged from 0.82 to 95%, with a mean ± SD of 48.14 ± 34.11, whereas the level of CD34 in ALL patients ranged from 0.8 to 98%, with a mean ± SD of 62.11 ± 36.78.
The expression level of HLA-DR in AML patients ranged from 0.4 to 94%, with a mean ± SD of 29.67 ± 26.8, whereas the level of HLA-DR in ALL patients ranged from 7.88 to 94%, with a mean ± SD of 54.42 ± 40.73. The expression level of CD3 in AML patients ranged from 0.6 to 12%, with a mean ± SD of 4.05 ± 4.33, whereas the level of CD3 in ALL patients ranged from 1.6 to 85%, with a mean ± SD of 27.58 ± 28.49. The expression level of CD7 in AML patients ranged from 0.01 to 14%, with a mean ± SD of 3.89 ± 4.98, whereas the level of CD7 in ALL patients ranged from 0.8 to 90%, with a mean ± SD of 29.01 ± 36.42. The expression level of CD10 in AML patients ranged from 0 to 2%, with a mean ± SD of 0.22 ± 0.63, whereas the level of CD10 in ALL patients ranged from 0.2 to 97%, with a mean ± SD of 35.46 ± 42.2. The expression level of CD13 in AML patients ranged from 0.2 to 52%, with a mean ± SD of 30.02 ± 14.91, whereas the level of CD13 in ALL patients ranged from 0.1 to 11.1%, with a mean ± SD of 3.28 ± 4.2. The expression level of CD19 in AML patients ranged from 0.01 to 9.3%, with a mean ± SD of 2.62 ± 3.55, whereas the level of CD19 in ALL patients ranged from 1.5 to 97%, with a mean ± SD of 53.12 ± 43.46.
The expression level of CD33 in AML patients ranged from 21 to 97%, with a mean ± SD of 39.9 ± 23.84, whereas the level of CD33 in ALL patients ranged from 0.61 to 37.9%, with a mean ± SD of 6.24 ± 11.28.
The expression level of CD14 in AML patients ranged from 0.03 to 42%, with a mean ± SD of 12.48 ± 15.87, whereas the level of CD14 in ALL patients ranged from 0.03 to 10%, with a mean ± SD of 1.38 ± 3.05. The expression level of CD235a in AML patients ranged from 0 to 35%, with a mean ± SD of 3.5 ± 11.07, whereas CD235a was not analyzed in ALL patients.
SALL4 expression in the studied groups
SALL4 mRNA was expressed in 14 (35%) cases of acute leukemia, it was undetectable in 26 (65%) cases ([Figure 4]), and was expressed in 10 (50%) samples of AML and in only four (20%) samples of ALL ([Figure 5]). SALL4 mRNA was undetectable in all cases of ITP (group II) (0%) ([Figure 6]) and in the control group (group III) (0%) ([Figure 7]) ([Table 4] and [Table 5]).
|Figure 4 SALL-4 expression in group Ia (acute myeloid leukemia): (a) SALL-4 mRNA expression in lanes 2, 3, 4, 5, and 7; lane 11 is the negative control and lane M is the DNA molecular size marker; (b) b-actin expression in the same samples.|
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|Figure 5 SALL-4 expression in group Ib (acute lymphoblastic leukemia): (a) SALL-4 mRNA expression in lanes 7 and 10; lane 11 is the negative control and lane M is the DNA molecular size marker; (b) b-actin expression in the same samples.|
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|Figure 6 SALL-4 expression in group II (immune thrombocytopenic purpura): (a) SALL-4 mRNA shows no expression; lane 11 is the negative control and lane M is the DNA molecular size marker; (b) b-actin expression in the same samples.|
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|Figure 7 SALL-4 expression in group III (control): (a) SALL-4 mRNA shows no expression; lane 11 is the negative control and lane M is the DNA molecular size marker; (b) b-actin expression in the same samples.|
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|Table 5 Sall-4 gene in acute myelobastic leukemia in comparison to immunophenotyping|
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There was no statistically significant difference between the expression level of CD markers and SALL4 in AML cases, whereas there was a statistically significant elevation of only the CD10 expression level in SALL4-positive ALL cases (P < 0.05) ([Table 4] and [Table 5]).
There was no statistically significant difference between the expression level of CD markers and SALL4 mRNA in AML cases, whereas there was a statistically significant reduction in the RBC count and the Hct and platelets level in SALL4-positive ALL cases (P < 0.05), and there was a statistically significant reduction in the platelet count in SALL4-positive AML cases.
| Discussion|| |
Acute leukemia is the most common malignancy in childhood. Its incidence is 4 and 0.7 cases of ALL and AML, respectively, per 100 00 children per year in 1-14-year-old children  .
Currently, 80% of the children with ALL treated in modern centers are alive and disease free at 5 years. The major contributors to this long-term survival are the improvements in anticancer therapies ,, .
In this study, it was found that acute leukemia is slightly higher in men than in women as 55% of the cases were male and 45% were female, with no statistical significant difference between them. Results similar to that of Ferlay et al.  were reported by other groups of researchers, as precursor B-lymphoblastic leukemia/lymphoma were expressing the antigens CD45, CD34, HLA-DR, CD19, CD10, and CD22 mainly. In precursor T-lymphoblastic leukemia/lymphoma, the antigens were mainly CD7, CD3, CD45, CD34, and HLA-DR , . Similar results were reported by other researchers such as Ling et al.  .
In the study by Hjalgrim et al.  , SALL4 mRNA was examined in cases of AML and ALL. Results were compared with ITP patients and normal control individuals , .
SALL4 mRNA expression was higher in cases of AML (50%) than in ALL (20%). In AML, the strongest expression was in AML-M1 (1/1) and AML-M2 (3/3) and to a lesser extent AML-M4 (1/2), whereas SALL4 mRNA was not detectable in AML-M5 and AML-M6. This is probably due to the fact that the leukemia samples had a variable range of leukemic myeloblast population (40-90%). There was no SALL4 mRNA expression detected in all cases of ITP patients and in normal control individuals  . Other results reported by other researchers such as Ma et al.  showed that SALL4 was constitutively expressed in all AML samples and failed to turn off in human primary AML, and the strongest expression was seen in AML-M1 and AML-M2, with no SALL4 expression detected in the normal bone marrow, the normal thymus, the normal spleen, and the normal peripheral blood. It was suggested that SALL4 was present in CD34+ HSCs/HPCs and was downregulated in mature granulocytes and lymphocytes. Also, it was expressed in myeloid leukemia cell lines. The leukemogenic potential of constitutive expression of SALL4 was tested in vivo by the generation of SALL4 transgenic mice. The transgenic mice exhibited dysregulated hematopoiesis, much like that of human myelodysplastic syndromes (MDS), and AML that was transplantable  . The MDS-like features in these SALL4 transgenic mice apparently did not require cooperating mutations and were observed as early as 2 months of age. The ineffective hematopoiesis observed in these mice was characterized, as it is in human MDS, by hypercellular bone marrow and paradoxic peripheral blood cytopenias (neutropenia and anemia) and dysplasia, which were probably secondary to the increased apoptosis noted in the bone marrow.
Shuai et al.  examined SALL4 expression levels in bone marrow mononuclear cells from MDS patients, AML patients, and normal control individuals using a semiquantitative RT-PCR, and found that higher levels of SALL4 expression were seen in MDS and AML samples than in control samples. As a result to the previous finding, the constitutive expression of SALL4 in leukemia may have prevented leukemic blasts from differentiating and/or gaining properties that were normally seen in HSCs, probably by interacting with additional mutations as leukemogenesis is a multistep pathology  . SALL4 mRNA expression in ALL cases was only 20%, as two out of 10 cases of ALL had detectable SALL4 mRNA. B-cell lymphoblastic leukemia showed the expression of SALL4 mRNA in two out of six (33%) cases, whereas no expression was detected in T-cell lymphoblastic leukemia (0/4). Other groups of researchers , .
A higher incidence of SALL4 mRNA expression was reported in seven out of eight B-cell lymphoblastic leukemia/lymphoma samples examined, which were positive for SALL4. Of the cell lines, only those of precursor B-cell lymphoblastic leukemia/lymphomas and AML were positive for SALL4. SALL4 mRNA was negative in all of the lymphoma cell lines that were derived from the precursor T-cell lymphoblastic leukemia/lymphomas. Hence, the expression of the SALL4 gene is restricted to CD34+ hematopoietic stem/progenitor cells during normal hematopoiesis, and it does not occur in normal mature hematopoietic cells and benign reactive lymphoid tissues , .
There was no statistically significant relationship between the levels of SALL4 mRNA expression and clinical data of the patients such as patients' age, sex, peripheral hemogram (except a significant reduction in the platelet count in SALL4-positive AML cases, which might be due to the progression of the severity of the disease and increased bone marrow encroachment by monoclonal blasts). In terms of the immunophenotyping of AML in this work, no statistically significant relationship was observed between SALL4 gene expression and all CD markers (CD34, CD45, HLA-DR, CD13, CD33, CD14, and CD235a). No statistically significant relationship was found between CD markers and SALL4 mRNA expression in ALL patients, except CD10, which increased significantly in SALL4 mRNA-positive leukemic patients, and this can be explained by the expression of SALL4 mRNA in B-cell lymphoblastic leukemia/lymphomas as discussed before; this may be explained by a common pathway shared by the leukemogenesis of precursor B-cell lymphoblastic leukemia/lymphomas and AML, and neither of them is related to T-cell lymphoblastic leukemia/lymphomas  .
| Conclusion and recommendation|| |
As the reduction of SALL4 has a drastic effect on the survival of leukemic cells, but not on normal ESCs as reported by other researchers, it is tempting to speculate that the SALL4/Bmi-1 regulatory pathway might be an attractive target for therapeutic intervention by inducing cancer stem cells to undergo apoptosis.
Further studies are recommended that study the expression of the SALL4 gene in different FAB subtypes with different types of blasts, which help in the detection of the significance of SALL4 expression and help in better understanding the role of the SALL4 oncogene in the process of leukemogenesis, focusing on SALL4 in various stem cell models, which may help one to address the fundamental connection between chromatin structure and cell function to develop new therapeutic strategies in the management of leukemia.
| Acknowledgements|| |
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]