|Year : 2020 | Volume
| Issue : 1 | Page : 35-39
Wilms’ tumor gene (WT1) expression levels as prognostic marker in pediatric acute lymphoblastic leukemia
Neveen L Mikhael1, Areej M Ibrahim1, Myriam A Helmy1, Hoda El Sheikh2
1 Department of Clinical Pathology, Alexandria Faculty of Medicine, Egypt
2 Department of Pediatric Oncology, Students Hospital, Alexandria, Egypt
|Date of Submission||22-Aug-2019|
|Date of Acceptance||04-Dec-2019|
|Date of Web Publication||10-Sep-2020|
Neveen L Mikhael
Department of Clinical Pathology, Alexandria Faculty of Medicine, Alexandria, 21131, Egypt
Source of Support: None, Conflict of Interest: None
Background Wilms’ tumor gene (WT1) encodes a transcription factor that has a role in kidney development and malignancy. WT1 is shown to be overexpressed in most adult acute myeloid leukemias and hence is an adverse prognostic factor. Its use as a prognostic marker in childhood acute lymphoblastic leukemia (ALL) is controversial.
Aim The aim was to study WT1 gene expression levels for diagnosis of ALL in a group of Egyptian children and to relate it to prognosis.
Participants and methods This study was conducted on 140 children newly diagnosed as having ALL; assessment of WT1 gene was done by real-time PCR in bone marrow (BM) samples at diagnosis and at day 28 of treatment.
Results WT1 gene expression was positive in 96 (68.6%) cases and negative in 44 (31.4%) cases. WT1 expression was related to aberrant expression of myeloid markers. There was a significant correlation between WT1 expression at diagnosis and at day 28 and minimal residual disease detected at day 28. No correlation was detected between outcome and WT1 level during follow-up.
Conclusion WT1 gene expression is related to response to therapy as defined by day 28 minimal residual disease.
Keywords: expression, pediatric acute lymphoblastic leukemia, Wilms’ tumor
|How to cite this article:|
Mikhael NL, Ibrahim AM, Helmy MA, Sheikh HE. Wilms’ tumor gene (WT1) expression levels as prognostic marker in pediatric acute lymphoblastic leukemia. Egypt J Haematol 2020;45:35-9
|How to cite this URL:|
Mikhael NL, Ibrahim AM, Helmy MA, Sheikh HE. Wilms’ tumor gene (WT1) expression levels as prognostic marker in pediatric acute lymphoblastic leukemia. Egypt J Haematol [serial online] 2020 [cited 2020 Oct 22];45:35-9. Available from: http://www.ehj.eg.net/text.asp?2020/45/1/35/294781
| Background|| |
Leukemia is the most common pediatric cancer, and its cure rates are increasing. It is the leading cause of disease-related death in childhood, and there has been improvements in its diagnosis and treatment ,. Minimal residual disease (MRD) testing is done to detect the efficacy of treatment protocol used. MRD is used to detect leukemic cells using flow cytometry and molecular markers in the abcsence of morphological evidence ,.
WT1 is an important regulatory molecule involved in cell growth and development. It is located at chromosome 11p13. It encodes for 10 exons and generates a 3 kb mRNA ,. WT1 gene encodes a transcription factor important for normal cellular development and cell survival. WT1 might have a regulatory role at the post-transcriptional level as well . It is expressed in a tissue-specific manner. In normal human bone marrow, WT1 is expressed at extremely low levels, mainly in primitive CD34+ population of cells .
WT1 is a expressed in a variety of malignancies, varying within different forms of human leukemia . The increased levels of WT1 can be found in both acute lymphoblastic leukemia (ALL) and acute myeloblastic leukemia (AML). Many studies have focused on WT1 expression levels in AML relating it to prognosis and even using it as a marker of MRD ,.
We examined WT1 mRNA expression levels in a group of children with newly diagnosed ALL and related it to ALL subtypes and clinical outcome.
| Participants and methods|| |
After Ethics Committee approval and informed consent, samples from 140 children with newly diagnosed ALL were tested for WT1 expression at diagnosis and after induction therapy. Patients were selected from two regional Tertiary Care Pediatric Hospitals and were all participant to the same treatment protocol. The female to male ratio of patients was 1.2 :░ the median age was 6 years (range: 1–18 years). They involved 96 patients with B-ALL and 44 patients with T-ALL. Characteristics of patients and samples are summarized in [Table 1].
|Table 1 Acute lymphoblastic leukemia characteristics of patients and samples|
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All patients in the study were treated using the Modified CCG protocol (1961) .
The Remission Induction phase (4 weeks of induction) uses vincristine intravenous (IV) (days 0, 7, 14, and 21), daunomycin IV (days 0, 7, 14, and 21), prednisone PO (days 0–27), l-asparaginase IM (3 x per week for nine doses total in induction), ara-C IT (day 0), and methotrexate IT (day 7 and also days 14 and 21 if leukemia seen in the spinal fluid). Day 14 bone marrow assessment is done; if blasts greater than 5%, PEG asparaginase IM, ara-C IV/SQ, cytoxin IV, and methotrexate IT are added. In the consolidation phase (5 weeks of consolidation), methotrexate IT (days 1, 8, 15, and 22), cyclophosphamide IV (days 0 and 14), ara-C IV/SQ (days 1–4, 8–11, 15–18, and 22–25), 6 MP PO daily, and prednisone PO (over 10 days weaning off) (plus radiation if CNS involvement) were used. For 8 weeks of interim maintenance, methotrexate IT (days 0 and 28), 6MP PO (days 0–41), and methotrexate PO (days 7, 14, 21, and 35) were uses. For 7 weeks of delayed intensification, vincristine IV (days 0, 7, 14), doxorubicin IV (days 0, 7, 14), l-asparaginase IM (3x per week for 6 doses), dexamethasone PO (days 0-20, then weaning off), cyclophosphamide IV (day 28), ara-C IV/SQ (days 29–32, 36–39), TG PO (days 28–41), and methotrexate IT (days 0, 28, 35) were used. In the maintenance phase (84-day courses), vincristine IV (days 0, 28, 56), prednisone PO (days 0–4, 28–32, 56–60), methotrexate PO (weekly, days 7–77), 6MP PO (days 0–83), and methotrexate IT (day 0, ±28) were used.
WT1 gene expression assay
Bone marrow samples were obtained at diagnosis and at day 28 of treatment. Bone marrow (BM) was examined for WT1 gene expression using RT-PCR. RNA was first isolated from the patients’ samples using Blood QIAamp RNA Blood Mini Kits (Qiagen, Germany). Reverse transcription was done on a thermal cycler (Applied Biosystems, USA), and the samples were stored at −80° until PCR amplification. Real-time quantitative detection of WT1 gene was done on Rotor Gene Q using ipsogen, WT1 ProfileQuant (QIAGEN GmbH). The level of WT1 gene was detected and expressed as a ratio to ABL gene (endogenous control) normally present in the human body.
Collected data were fed to the computer and analyzed using IBM SPSS software package version 20.0 (USA). Qualitative data were described using number and percentage. Quantitative data were described using range, mean, SD, and median. Significance of the obtained results was judged at the 5% level. Log-rank test were used to assess survival.
| Results|| |
WT1 expression levels
The WT1/ABL ratio was measured in all cases of ALL in the study and divided to high WT1 level (WT1/ABL ratio >0.44), intermediate WT1 level (WT1/ABL ratio 0.44–0.0003), and low WT1 level (WT1/ABL ratio <0.0003). High levels of WT1 were detected in 48 (34.3%) patients, intermediate levels in 48 (34.3%) patients, and low levels in 44 (31.4%) patients. Day 28 levels were either intermediate in 16 (11.4%) patients, low in 32 (22.8%) patients, or undetectable in 92 (65.8%) patients.
WT1 levels and disease characteristics
WT1 level did not correlate with age, sex, hemoglobin concentration, leukocyte count, or blast count at diagnosis. We also observed no significant difference between WT1 level and ALL type. However, pro-B-ALL expressed high level of WT1 but did without statistical significance. WT1 expression level was significantly higher in patients with ALL with aberrant expression of myeloid markers (P=0.028).
WT1 levels and minimal residual disease
Clinical outcome data were available for all patients in the study. WT1 expression level at diagnosis was correlated with MRD at day 14 and MRD at day 28 (end of induction). Negative MRD at day 28 showed significantly lower levels of WT1 expression at diagnosis (P=0.029).WT1 level was re-measured at the end of induction and correlated to MRD at day 28. Significantly lower WT1 level after induction was seen in patients with negative MRD at day 28 (P=0.021) ([Table 2]).
|Table 2 Relationship between WT1 level and minimal residual disease at day 28|
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WT1 level and patients’ outcome
Patients were followed up for 15 months. The outcome of patients was complete remission, partial remission, relapse, or death. Both patients with partial remission and relapse were considered as cases with remission failure. Relapsed patients were mostly those with high WT1 levels at induction; however, there was no statistical significant relation between outcome of treatment and WT1 level done at diagnosis (P=0.122) or at the end of induction (P=0.332).
Kaplan–Meier curves were drawn showing overall survival and disease-free survival. Log-rank test was done to detect significance of WT1 level on DFS and OS. The DFS curve was drawn with n=132, as eight cases died before the continuation of the consolidation phase. It shows that the relapse occurs more in the patients with high WT1 levels, but this did not reach statistical significance (P=0.178) ([Figure 1]).
| Discussion|| |
Wilms tumor gene expression has been regarded as a prognostic tool in acute leukemia, especially AML. Our study aimed at studying WT1 gene expression in a group of patients with childhood ALL.
In this study, WT1 expression was found in almost 75% of cases of ALL. These percentages were similar to those obtained in previously published research on pediatric ALL ,. However, other studies reported percentages as low as 55%  or even 14% .
For patients with ALL in the present study, WT1 expression was not affected by age or sex. Studies have shown differences in different age groups ,. A study found significantly higher expression in males , whereas other studies did not report any difference in expression between the two sexes ,. However, it is difficult to compare the results owing to the diversity in number of patients present in each age group within the studies.
There was no significant difference between the WT1 level and the clinical presentation and laboratory data at the time of diagnosis. This is in agreement with other studies which did not find a correlation between WT1 expression level and clinical presentation, white blood cells, platelet, hemoglobin level, and blast cell count ,,.
Regarding the immunophenotypic subtypes of ALL, WT1 expression levels did not differ between B-ALL and T-ALL, which is similar to a study by Ibrahim et al.  Higher levels of WT1 in T-ALL compared with B-ALL were detected in different studies ,,. The immature subtype of B-ALL (Pro-B-ALL) showed higher WT1 levels but did not reach statistical significance. Similarly, two previous studies showed that the more mature the B-ALL, the more similar the WT1 expression level to the normal bone marrow background ,.
Moreover, ALL blasts with aberrant co-expression (CD13, CD33, or both) were associated with significantly higher levels of WT1. These results were similar to Yoon et al.  who found that blasts with aberrant myeloid markers had higher WT1 expression levels than blasts without co-expression of myeloid markers. They explained that this corresponds to the overall difference in WT1 expression level between AML and ALL.
Data on using WT1 as a marker of MRD are little and controversial. In our study, positive MRD was significantly related to higher WT1 levels at diagnosis and higher levels after induction therapy.
Zhang et al.  found that WT1 is unreliable to predict relapse in ALL, and there was no statistical difference between the levels of WT1 at diagnosis and after chemotherapy, so an optimal value of cutoff was not found.
In the present work, high WT1 expression levels was correlated with MRD. Accordingly, a study  found a higher risk of relapse with WT1 gene overexpressed. Another two studies showed that rapid decrease of WT1 expression level predicted a good response to the induction therapy, and low expression of WT1 correlates with remission status. This is unlike the study by Yoon et al.  which did not find any association between WT1 expression level and remission rate or relapse rate and showed that the prognostic role of WT1 in ALL is limited. ,.
WT1 level was re-measured after induction treatment. Higher levels of expression WT1 at diagnosis were associated with significantly higher levels of WT1 after induction and significantly positive MRD. This is in contradiction to Hagag et al.  who found that high levels of WT1 have negative MRD after therapy.
Induction chemotherapy has led to decrease in the level of WT1 in most patients. There were no patients with high levels of WT1 after induction therapy, and 83% of the patients showed negative or low WT1 expression levels. This result is similar to Horvath et al.  who found that chemotherapy as short as 1 month has led to undetectable WT1 gene levels in most patients.In the present work, patients were followed up for a median of 15 months, and the influence of WT1 expression levels on the OS and DFS was not significant. This is consistent with studies from Ibrahim et al. , Magyarosy et al.  and Boublikova et al.  who reported that higher levels of WT1 were not associated with longer DFS. On the contrary, Hashii et al.  tested WT1 expression in 22 patients with ALL at diagnosis, at the end of induction, and after second consolidation chemotherapy and found that patients who were negative or with low WT1 gene at these time points had significantly better DFS and OS than those with high WT1 level.
Our results provide evidence that WT1 is expressed at very high levels in pediatric ALL and is mainly related to MRD positivity. Weaknesses of the study are the fact that we did not correlate WT1 with cytogenetic and molecular data as stated by a study by Uckun et al. .
| Conclusion|| |
In conclusion, WT1 overexpression can be found in almost all cases of ALL, especially immature types. Therefore, WT1 may be used as a potential molecular marker for monitoring of clinical progress and response to treatment. It can be used as a MRD combined with flow cytometry. However, WT1 expression at diagnosis has no prognostic effect on the clinical outcome in childhood ALL.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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