STUDY OF ALTERNATIVE WILMS TUMOR GENE EXPRESSION IN ACUTE MYELOID LEUKEMIA

Magdy Mamdouh El Bordiny 1 , Ashraf Hussien Al-Ghandour 2 , Reham Abdel Haleem Abo Elwafa 3 and Omneya Mohamed Kamal Fayed 4 . 1. Professor in Clinical Pathology Department, Faculty of Medicine, Alexandria University, Alexandria, Egypt. 2. Professor in Internal Medicine Department, Faculty of Medicine, Alexandria University, Alexandria, Egypt. 3. Lecturer in Clinical Pathology Department, Faculty of medicine, Alexandria University, Alexandria, Egypt. 4. Specialist in, Alexandria University Hospitals, Alexandria, Egypt. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History


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Although the oncogenic behavior of WT1 in leukemia has been proved, the mechanism has not yet been clearly explained.
In humans, the WT1 gene is located on chromosome 11p13 and consists of 10 exons. Many different isoforms exits for WT1, and it is estimated that it can have over 36 different isoforms generated by alternative transcription initiation, mRNA splicing and alternative translation initiation. (6) Recently an alternative promoter incorporating a unique first exon has been described. The alternative WT1 transcript (AWT1) encodes a 32 kDa protein deficient for the first 147 amino acids of the N-terminal of the WT1 protein, which lacks the repressor domain and the RNA recognition motif of the full-length protein. (7,8).
The Wilms' tumor gene 1 (WT1) was first reported as a tumor suppressor gene in Wilms' tumor. However, later studies have shown the oncogenic properties of WT1 in a variety of tumors. It was recently proposed that WT1and its isoforms are chameleon genes, due to its dual functions in tumorgenesis. (9,10). This may be used as a potential molecular marker for diagnosis, monitoring the clinical progress, the response to treatment, as well as a target for the development of novel therapeutic approaches. (11,12)

Subjects and methods:-
The present study was conducted in Clinical Pathology Department Faculty of Medicine, Alexandria University, Egypt. Informed consent was obtained from patients and controls according to the Ethical Committee for Human Research in the Alexandria UniversityHospitals.
Samples were collected from forty patients with newly diagnosed AML aged from 25 to 78 years admitted to the Department of hematology, Elmiry Hospital between August 2015 And June 2016.The diagnosis was made by standard morphological analysis and flow cytometryon BM/PB samples at diagnosis. Forty healthy subjects with matched age and sex and with no knownmalignancy were used as control group.
The aim of the present work is to assess the expression pattern of alternative AWT1 gene and to determine if this expression profile has correlation with age ,sex.haemoglobin level,WBCs count ,blast countand AML subtypes. Reactions were incubated at 95 o C for 10 minsfollowed by 45 cycles of 95 o C for 10s, 60 o C for 15s and 72oC for 25s. Beta actin was used as endogenous reference for normalizing the expression levels of AWT1 gene.

Detection of degree of expression of AWT1 in cases and controls:
Regarding the level of AWT1expression at initial diagnosis we found that it was significantly overexpressed in AML patients relative to control group With a median of 5.54 in AML cases compared to 0.76 in control group (p<0.001). (Figure 1) 1051 7.5% -- Figure 1:-Comparison between the two studied groups according to AWT1 Expression.
We found no significant correlation between age, sex and expression of AWT1,also we found no correlation between hemoglobin level and gene expression (P=-0.532.r = -0.11), while there was a positive correlation between white blood cells count and blast count and AWT1 expression (p= 0.10, 0.001) (r=0.40, 0.060) respectively.
Also, we found a positive correlation between AML subtypes and AWT1 expression where AWT1 was highly expressed in M3 followed by M4 (74.7% and 68.9% respectively).

Discussion:-
Wilms' tumor gene 1 (WT1) is gaining increasing attention as a therapeutic target molecule due to its common expression in acute leukemias and its involvement in cell proliferation. (13) AWT1 maintains WT1 exonic structure between exons 2 and 10, but deploys a new 5' exon located in intron 1 of WT1. The AWT1 gene predicts proteins of approximately 32kDa, comprising all exon 5 and exon 9 splicing variants previously characterized for WT1. (14) While we found the levels of WT1 expression were significantly different between different AML subtypes (P=0.001), Bergmann et al (15) found no relationship concerning the FAB subtype of AML and WT1 expression except in M5 AMLs, which expressed WT1 mRNA in only lower percent of the patients versus in all other de novo AML. This difference may be due to different samples characteristics as we used De novo cases only while they used de novo, MDS related and relapsed cases.
Our study revealed that the AWT1 gene was found to be significantly overexpressed in AML patients relative to control group as AWT1 was overexpressed in 100% of our patients compared to 22% of controls (P˂0.001).
Our results are consistent with the findings of Guillaumet-Adkins et al. (16) who used qRT-PCR on a wide range of leukemia and lymphoma cell lines to evaluate the transcription levels of the WT1, AWT1 and the non-coding antisense transcript WT1-AS. In all myeloid origin cell lines evaluated the WT1 and AWT1 transcripts were readily detectable, with high correlation, indicating that they are likely to be co-regulated. The WT1-AS transcript was also expressed in most of the myeloid cell lines. In contrast, the majority of B, T cell leukemic cell lines and lymphomas did not express these transcripts. Moreover, western blotting using an antibody directed against the C-terminal of the WT1 protein revealed that the abundant RNA levels for WT1 and AWT1 isoforms are translated into nuclear retained proteins in the myeloid derived cell lines, consistent with their transcription factor function.
The same results were reported by Furuhata et al. (17) who illustrates the relative mRNA levels of WT1 from 23 AML and 3 ALL patient bone marrows. Most leukemia patients (85%) showed WT1 mRNA levels of more than 10^2 of GAPDH mRNA, whereas the absence of WT1 mRNA was observed only in one AML patient. Additionally, they studied the WT1 mRNA levels in both normal human hematopoietic stem cell fractions (CD34‫‬ +, CD38-) and progenitor cell fractions (CD34+‫‬ , CD38‫‬ +). WT1 mRNA levels of normal stem cell and progenitor cell fractions were significantly lower than those of AML samples with high WT1 mRNA.
In Bergmann et al (15) study mRNA expression was found in in 77% of all 161 studied samples. The frequency of WT1 expression between de novo AML and AML with antecedent MDS did not differ. Only in patients in first relapse a slightly higher incidence of WT1 mRNA (87.5% v 74.6%) was found.
Moreover, the Overexpression of WT1 in AML have been made by studies in human leukemia cell lines and murine models. Notably, downregulation of WT1 expression in primary AML and CML cases (chronic phase and blast crisis) resulted in inhibition of cell growth.In order to further define the role of WT1 overexpression in leukemogenesis, a transgenic murine model of WT1 overexpression in the hematopoietic system was established and bone marrow from transgenic mice overexpressing WT1 was transduced. By contrast, it led to rapid onset leukemia, with a median time to leukemia development of 50 days. (18) These results were supported by the results of Yang et al. (19) When the levels of WT1 expression levels were studied using microarray in AML patients the majority of patients have high levels of WT1. However, some subgroups, have low to absent levels of WT1. In these patients, high levels of WT1 expression may not be well tolerated. This possibility is supported by the growth inhibiting effect of WT1. For example, when the WT1 KTS(+) isoform is expressed in a WT1-negative human myeloblastic leukemic cell line, M1, decreased tumor formation is observed when the cells are injected into immunocompromised mice. This is consistent with a tumor suppressor function of WT1 in some forms of AML.
Interestingly, a study was carried out to determine the prognostic relevance of WT1 gene expression levels in both BM and PB samples with regard to overall survival (OS) and freedom from relapse (FFR) during follow up. They analyzed the effects of a ≥1-log reduction and a ≥2-log reduction in WT1 mRNA expression. They found that the achievement of ≥1-log reduction in BM, WT1 was associated with improved OS and FFR. (20) 1053 Despite the limited number of patients in this study, their observations were consistent with previous studies, which showed that early MRD measurements could provide predictive information on patient outcome.
AWT1 expression analysis is a promising markers for diagnosis and MRD assessment in AML but larger patient numbers and more studies would be necessary to confirm these data.