Wilms' tumor is common in children and is caused by Wilms' tumor gene 1 (WT1). The WT1 mutation has been reported in a variety of hematologic malignancies. Changing expression of miRNA molecules has also been shown to play a role in the development of Wilms' tumor. Considering the fact that WT1 can be used as a clinical biomarker in leukemia cases, it can be a basis for immunotherapy of leukemia. WT1 is a gene that can be used as a prognostic biomarker for minimal residual disease, as well as the detection of relapse for clinical remission in leukemia. Furthermore, it can be considered as a predictive biomarker for the treatment of leukemic patients after allogeneic transplantation. This study aimed to review WT1 expression in leukemia, its involvement in miRNAs expression, as well as its importance in prognosis and treatment of leukemia.

Introduction

Wilms' tumor gene 1 (WT1) chromosome (11p13) is essentially a tumor suppressor gene[1] responsible for the development of Wilms' tumor (a type of pediatric kidney cancer), as well as other syndromes such as WAGR (Wilms' tumor, aniridia, genitourinary anomalies, and mental retardation) and Denys–Drash syndrome (DDS).[2],[3] In contrast to other tumor suppressor genes such as p53 or RB1, the normal expression of WT1 in adults is limited to a few tissues, including genitourinary system. In bone marrow (BM), the expression of WT1 in normal primitive progenitor cells is at a low level; however, the overexpression of WT1 mRNA is observed in several solid tumors and hematologic malignancies such as acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), chronic myeloid leukemia (CML), and myelodysplastic syndromes (MDS).[4],[5] Therefore, WT1 expression can be a universal molecular biomarker in malignant hematopoiesis, and WT1 mRNA monitoring in peripheral blood (PB) and BM has been reported to determine minimal residual disease (MRD) and relapse in leukemias.[1],[4] In addition, some studies have shown that evoking anti-WT1 immune response using WT1 peptide vaccine may induce tumor regression in a number of patients.[1]

Although the biological significance of WT1 expression in leukemia patients has not been elucidated, studies indicate that WT1 can contribute to the pathogenesis of human leukemia due to its role in the inhibition of transcription, as well as involvement in cellular differentiation and growth arrest. Other studies also suggest that the prognosis in leukemia patients can be inversely associated with the expression level of WT1.[4] The purpose of this review is to discuss the role of WT1 as a detectable tumor biomarker for prognosis and its importance for immunotherapy and prediction of treatment in hematologic disorders.

The Structure and Function of Wilms' Tumor Gene 1

WT1 gene is normally expressed in mesodermal tissues during embryogenesis and encodes a zinc-finger transcription factor.[6] It was first detected as a tumor suppressor gene in patients with WAGR and was subsequently reported as a mutational target in 10%–15% of sporadic cases of Wilms' tumor, as well as in WT1-associated DDS and Frasier syndrome. WT1 is initially expressed in the cells and tissues progressing toward genitourinary and hematopoietic systems. The expression level of WT1 is inversely related to differentiation in both systems, and its mutation causes Wilms' tumor, a common type of renal malignancy in children. WT1 expression in hematopoietic system is observed in CD34+ progenitor cells but not in mature leukocytes. In addition, WT1 is expressed in high levels in acute leukemia cells, and it may also be involved in leukemogenesis. WT1 could be involved in cell survival, proliferation, and differentiation both as tumor suppressor and oncogene, although its role in normal and malignant hematopoiesis is still unknown. These different functions can be linked to downstream targets, as well as the expression of different WT1 isoforms. There are four main isoforms of WT1, which are a function of two primary splice variants, including the splicing of exon 5 and 9 nucleotides encoding three amino acids between exons 9 and 10 (KTS lysine [K], threonine [T], and serine [S]).[7] WT1 is first expressed in cells and tissues differentiating into genitourinary and hematopoietic systems.[8],[9] WT1 protein has a proline-glutamine-rich N-terminal transcriptional regulatory domain (exons 1–6) and four C-terminal zinc-finger domains (exons 7–10) that facilitate its binding to DNA. Exons 5 and 9 are subject to alternative splicing, which results in the formation of four different splice isoforms,[10] including 17AA+/KTS+, 17AA+/KTS−, 17AA−/KTS+, and 17AA − KTS−, as well as other isoforms.[9] Depending on the expression level, specific isoform, and cellular context, WT1 protein can act as a transcription activator or repressor.[11] In addition, this protein is essential for cell proliferation and differentiation and is involved in the inhibition of apoptosis along with p53 and bcl2.[12]

WT1 is involved in the regulation of cell growth and differentiation. The nuclear and transcriptional activity of WT1 has been observed to increase in most cases of acute leukemia. The expression level of WT1 is associated with the presence, persistence, or reappearance of leukemic hematopoiesis, which could raise it as a strong prognostic factor.[8]

Expression and Alternation of Wilms' Tumor Gene 1 in Leukemia

Acute myeloid leukemia

AML forms a class of myeloid disorders characterized by increasing number of myeloid-derived blast cells, cell differentiation arrest, and accumulation of undifferentiated cells in BM.[13],[14] AML is a heterogeneous disease clinically and genetically, which accounts for 15%–20% of leukemia types in children. The majority of AML patients harbor at least a chromosomal abnormality in BM at diagnosis. However, 40%–49% of adult patients and 25% of children with AML are devoid of any chromosomal abnormality, which is known as cytogenetically normal AML cases (CN-AML) of predominantly adult patients.[8],[15] Multiple genetic abnormalities have been observed in this group of patients with a prognostic value. These abnormalities include mutations in FLT3, NPM1, CEBPA, and MLL genes, as well as the aberrant expression of BAALC, ERG, and MN1 genes.[15] Identification of these markers may be effective to predict the response of patients to treatment and their survival rate.[16]

WT1 is an essential molecular marker in biology and treatment in this group of patients.[2] WT1 mutation is known to be associated with increased proliferation of AML blast cells, as well as poor prognosis in patients through increasing expression of ERG and BAALC genes.[17] WT1 may act as a tumor suppressor gene or an oncogene.[2] However, the expression of different WT1 isoforms and their target genes may vary in different cells. WT1 expression has been reported in 70%–100% of AML patients, and its mutation is seen in 10%–15% of adults with AML. Mutation and increased expression of WT1 can interfere with survival and relapse rates. In addition, the assessment of WT1 expression is a marker of MRD after treatment.[16] The overexpression of WT1 has been reported in AML subtypes, which is independent of WT1 mutation that occurs in 10% of cases. Studies on the role of WT1 expression as a marker of MRD show that WT1 has a low expression level in normal BM, which is increased in the majority of AML patients at diagnosis, returning to baseline following effective treatment and increasing in cases of relapse.[18] WT1 gene mutations in exons 7 and 9 occur in 9%–13% of AML patients,[19] which are reported to be associated with lower complete remission, higher relapse rates, shorter disease-free survival (DFS), and overall survival (OS) but have no prognostic value compared with wild-type WT1 (WT1 wt).[20]

It has been recognized that the polymorphism of WT1 gene, as well as its mutation, can be considered as a potential prognostic factor in AML subtypes, but this has not been confirmed by all the studies. In this regard, rs16754 WT1 gene polymorphism in AML patients undergoing chemotherapy caused an increase in OS and DFS, while complete remission in these patients was not affected by WT1.[21] In addition, SNP rs16754 in exon 7 of WT1 gene in CN-AML patients was associated with shorter survival. Therefore, WT1 gene mutations can be considered to predict the poor prognosis in CN-AML patients.[22] Since WT1 mutations and polymorphisms can be used as prognostic biomarkers in AML patients,[21],[22] it is suggested to assess WT1 along with other prognostic biomarkers in these patients [Table 1].{Table 1}

Acute lymphoblastic leukemia

This is the most common childhood malignancy characterized by monoclonal expansion of immature T and B lymphoid precursors in the BM, PB, and organs of the body.[23],[24] Increased expression of WT1 has been reported in the majority of these patients. For example, Tosello et al. reported that the prevalence of WT1 mutations in adult and pediatric T-lineage ALL patients was approximately 12 and 13%, respectively, and the majority of their mutations were similar to AML patients (frameshift mutations within exon 7).[5],[25] Furthermore, Chiusa et al. studied ALL patients and showed that contrary to previous reports concerning increased WT1 gene expression in 44%–86% of ALL patients, WT1 expression was increased in almost all adults with ALL. In addition, this study showed that the level of WT1 expression correlated with the survival of patients and could be considered a prognostic factor in these patients.[19] Studies have shown that the expression levels of WT1 are significantly higher in children with T-ALL compared to children with B-cell precursor-ALL but are lower than its normal levels in BM of these patients. Furthermore, in relapsed childhood ALL cases, abnormally high or low expression levels of WT1 have been observed.[26] Therefore, in general, it can be stated that the expression levels of WT1 in children with ALL are highly variable and lower than its expression levels in patients with AML and adult ALL. The varying expression of WT1 may indicate that WT1 expression in these patients cannot be regarded as a useful marker for MRD, but the very low expression of it in children with ALL compared to its normal physiological expression levels in BM can raise WT1 as a prognostic factor of relapse in children with ALL.[27] In addition to cytogenetically normal cases of ALL, the variation of WT1 expression in cytogenetically abnormal ALL subtypes has also been examined. Evidence suggests that high levels of WT1 expression in patients with MLL-AF4 and B-cell receptor (BCR)-ABL p190 translocation can be considered as a prognostic biomarker in ALL patients harboring these two translocations.[5]

Myelodysplastic syndromes

MDS is a clonal disorder characterized by the abnormal maturation and differentiation of myeloid precursors and increased resistance to proapoptotic signals.[28],[29] Studies indicate that WT1 is a likely tumor suppressor gene and a tumor marker for leukemic or preleukemic MDS blast cells. In this regard, it has been found that the expression levels of WT1 are indicative of MDS progression, in such a way that WT1 expression is increased with disease progress.[30] In addition, pancytopenia is among clinical complications developing following hematologic and nonhematologic diseases. The high expression of WT1 is a prognostic biomarker of pancytopenia for MDS and even AML among other hematologic malignancies.[31] Clinical evidence has indicated that a single dose of WT1 vaccination in patients with leukemia due to MDS is effective in the reduction of leukemic blast cells in addition to increasing WT1-specific cytotoxic T-lymphocytes. Thus, according to clinical evidence, WT1 peptide can be a basis for immunotherapy of MDS patients.[32]

Chronic myeloid leukemia

This is a clonal stem cell disorder associated with increasing number of myeloid cells in BM and their accumulation in PB. Reciprocal translocation between chromosomes 9 and 22 (known as Philadelphia chromosome) that generates the BCR-ABL fusion protein is typical of this type of leukemia.[33] Although the expression of WT1 is increased in CML, no significant correlation has been observed between WT1 with BCR-ABL in newly diagnosed CML patients. Therefore, WT1 has not been suggested as a marker for monitoring of CML patients.[34] However, other studies have shown that despite the overexpression of WT1 mRNA and protein in CML cell line, the use of WT1 inhibitors could inhibit the proliferation of CML cells in cell cycle and reduce the transcription of WT1 and P210 genes. It could be argued that the WT1 gene is a therapeutic target for Ph+ leukemia patients.[35]

Other

Chronic lymphocytic leukemia (CLL) is the most common type of leukemia in middle age that is associated with an abnormal proliferation of lymphocytes and their resistance to apoptosis.[36] 60%–65% of CLL cases harbor a mutation in the variable region of immunoglobulin heavy chain (IGHV), which can alter BCR affinity to antigen, but 35%–40% of CLL cases lack IGHV mutations.[37],[38] Mutation in IGHV gene is suggested as a marker of favorable prognosis, while the unmutated CLL (U-CLL) is more aggressive than the mutant case (M-CLL).[37],[39] Monitoring of patients with leukemia, especially multiple myeloma (MM), during and after therapy is of particular importance to assess MRD to determine the effectiveness of treatment. Despite the fact that the expression levels of WT1 as a marker of MRD are increased in MDS patients, evidence suggests that the expression of WT1 mRNA is normal in newly diagnosed MM patients. Therefore, it can be concluded that WT1 is not a prognostic biomarker in MM patients [Table 1].[40]

Impact of Wilms' Tumor Gene 1 on miRNAs Expression

miRNAs form a group of small noncoding RNA molecules inhibiting their target mRNAs by binding the 3-UTR region. Studies have shown that miRNAs play an important role in the development of WT1, as well as their involvement in the kidney development.[48] The oncogenic role of WT1 in leukemogenesis has been observed, as well as its increased expression during leukemia. However, some studies have shown that miRNAs such as miR-125a can regulate and suppresses WT1 expression by the 3'UTR of WT1 mRNA in myeloproliferative disease.[49] Table below shows different roles of miRNA molecules and their altered expressions in WT1 [Table 2].{Table 2}

Wilms' Tumor Gene 1 as a Predictive Biomarker in Leukemia

Several leukemias cannot be cured with available treatments, including MDS and acute leukemias. In addition, the efficiency of donor leukocyte infusion in leukemia patients is restricted for reasons such as myelosuppression and graft-versus-host disease; therefore, the need for new therapeutic strategies arises in this context.[42] Leukemic patients who cannot respond to treatment receive allogeneic hematopoietic stem cell transplantation (HSCT). Although the prognosis of leukemic patients with HSCT have been improved, relapse remains as a major challenge in these patients.[54]

WT1 is among the genes found to be applicable as a prognostic marker to detect relapse in clinical remission in AML and ALL cases.[55] WT1 is an interesting target in the field of immunotherapy in leukemia. Immunological activity of WT1, as well as its specific antibodies and cytotoxic T-lymphocyte responses have been detected in different hematologic malignancies.[56] Targeting WT1 in acute leukemias for immunotherapy has been suggested in some studies due to its high concentrations in leukemic cells. On the other hand, the use of dendritic cells and the generation of WT1-specific CTL have been shown effective in the development of immune cells.[57]

Studies have shown that WT1 peptide vaccine has been effective in leukemia patients, and clinical improvement has been observed in different types of patients with hematological malignancies.[58] WT1 is an active immunogenic antigen, and IgG and IgM antibodies for WT1 have been found in leukemic patients.[59] In allogeneic HSCT, graft-versus-leukemia (GVL) can occur, in which the leukemia cells can be removed by donor T-cells. Therefore, the GVL effect may be increased with WT1 peptide vaccine.[54] WT1-derived CD8 T-cell epitopes have been investigated in some studies. HLA-A0201-restricted peptide 124-134, HLA-A24-restricted peptide 235–243, WT1-235 (CMTWNQMNL), and its analog (CYTWNQMNL) peptides can cause CTLs to lyse WT1þ leukemic cells in a HLA-A24-restricted manner.[60],[61] WT1-derived CD4 epitopes also have been found to be able to bind HLA-DR types in some studies. Peptide 124–138, 247–261, 332–347, and 337–347 bind to HLA-DR53, DRB1*0401, HLA-DR53, HLA-DRB1*0405, and HLA-DP5, respectively.[62],[63]

In conclusion, WT1 peptide vaccine treatment can be considered as a favorable tool to improve leukemic patients with new peptides and their analogs under study so that WT1 could be suggested as a predictive biomarker for usefulness of treatment after allogeneic HSCT.

Conclusions and Future Perspectives

A number of cytogenetic abnormalities observed in leukemia have been detected in WT1, including chromosome 22 deletion. Perhaps, these abnormalities are indicative of a good or poor prognosis in leukemia in case of further investigations given their favorable or poor prognosis in WT1.[64] On the other hand, preventing relapse after allogeneic HSCT with WT1 peptide vaccine is an interesting treatment strategy in the future, which can introduce WT1 as a predictive biomarker for leukemic patients; however, further studies are required to achieve this goal.

Acknowledgment

We wish to thank all our colleagues in Golestan Hospital clinical research development unit, Ahvaz Jundishapur University of Medical Sciences.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.