The survival rate of pediatric patients with acute myeloid leukemia (AML) approached 70%, with approximately 30% experiencing relapse, which is the main cause of treatment failure (1). Measurable residual disease (MRD) is an important biomarker in AML patients used for prognosis and response assessments (2).

The prognostic value of MRD detected by real-time quantitative polymerase chain reaction (RT-qPCR) or multiparametric flow cytometry (MFC) is well established, and relapse occurs more likely in patients with detectable MRD (3). Previously published data suggested that patients with one positive and one negative MRD results performed by two different techniques have higher relapse risk than patients with two negative MRD results, but lower relapse risk than patients with two positive MRD (2, 4, 5).

Specific genetic aberrations, such as RUNX1::RUNX1T1, CBFB::MYH11, and nucleophosmin 1 (NPM1) mutations, constitute markers of MRD but about 40% of children with AML harbor leukemia-specific targets, which makes these genetic targets clinically applicable in only minor fraction of children (6, 7). Therefore, it was crucial to identify other molecular targets applicable to the majority of patients.

Wilms tumor gene 1 (WT1) is overexpressed in approximately 80% of children with AML. Previous cohort studies in children and adults evaluated WT1 gene overexpression at diagnosis, during treatment, and pre-, and post-hematopoietic stem cell transplantation (HSCT) to provide a target for novel immunotherapeutic approaches and to advocate it as a universal marker for MRD assessment.

The European Leukemia Network researchers (ELN) validated a quantitative WT1 assay and established reference ranges for WT1 expression in peripheral blood (PB) and bone marrow (BM) by analyzing a large number of control samples. This study showed that (≥2 log) reduction in WT1 transcript after induction predicted reduced relapse risk, also failure to reduce WT1 transcripts below this threshold limits after intensification predicted increased risk of relapse (p=.004) (8). For pediatric AML, Lapillonne et al. observed that WT1 level higher than 50 × 10⁴ after induction was an independent prognostic risk factor of relapse (p=.002) and death (p=.02) (9).

A recent consensus from the ELN MRD working group recommends WT1 as an important tool in monitoring MRD and stratifying patients with AML especially those who lack a more sensitive marker and thus could influence treatment strategy (2).

The objective of this study was to assess the prevalence of WT1 gene overexpression in pediatric patients with AML treated at Children’s Cancer Hospital Egypt (CCHE-57357) on AML protocol adopted from the Children Oncology Group (COG) (NCT01371981) (10) and to evaluate the prognostic significance of WT1 as an MRD marker on survival and disease outcome.

This study comprised 163 pediatric patients with De novo AML diagnoses who received therapy at CCHE-57357 between January 2019 and May 2020. A minimum follow-up period of one year from the completion of treatment was ensured for all participants. The study was approved by the Institutional Review Board (IRB), and informed consent was secured prior to the start of therapy.

Patients with conditions including acute promyelocytic leukemia (APL), Down syndrome myeloid neoplasm, myelodysplastic syndrome (MDS), therapy-related AML (t-AML), and myeloid sarcoma were excluded from the study. Data related to the patients were gathered from their electronic medical records and initial disease assessments included morphology, immune-phenotyping, cytogenetics, and molecular studies. For disease evaluation bone marrow aspirate (BMA) was done after each cycle and MRD was monitored by MFC based on the ELN proposed consensus using 8-10 colors monoclonal antibody (mAbs) panels. We adopted two methodologies: the Leukemia Associated Immunophenotype (LAIP) approach and the Different from Normal (DFN) approach. MRD is detected based on LAIP’s present on any population and any deviation seen from normal patterns (7, 11). For patients with core binding fusion transcript (CBF), molecular MRD assessment by PCR was done post-intensification I.

Patients were enrolled on the CCHE 57357 AML protocol, which was adapted from the modified COG protocol (NCT01371981) (10) and based on their cytogenetics and molecular abnormalities, patients were categorized into Low risk (LR) groups: Patients with favorable cytogenetics, including. CBF (t(8;21) (q22;q22.1); RUNX1::RUNX1T1, inv (16) (p13.1;q22) or t (16;16) (p13.1;q22); CBFB::MYH11), Nucleophosmin 1 (NPM1), and CEBPA abnormalities. Intermediate risk (IR): Patients without either favorable or unfavorable criteria. High risk (HR): Patients with unfavorable cytogenetics, such as monosomy 7, monosomy 5, complex karyotype (more than three chromosomal aberrations), and FLT3 internal tandem duplications (FLT3/ITD) with a high allelic ratio (> 0.4, FLT3/ITD positive). Additionally, IR patients were further classified into HR and LR categories based on MRD response measured by MFC after the first induction. LR was defined as MRD <0.1% (10).

All patients received 4 cycles of chemotherapy and only high-risk were offered allogeneic hematopoietic stem cell transplantation (Allo-HSCT) in complete remission (CR) if a matched sibling donor is available. The chemotherapy regimen details and dosages are given in “ Supplementary Table 1 ”.

The patients’ characteristics were compared using the chi-square test and Mann-Whitney test with Bonferroni multiple hypothesis P-value adjustment. Overall, event-free, and relapse-free survival probabilities (OS, EFS, RFS) were calculated using Kaplan-Meier analysis, and comparisons between patients were performed using the log-rank test. OS was defined as the interval from the date of diagnosis until the date of death or last contact date. EFS was delineated as the interval from the date of diagnosis until the occurrence of an event, which could be relapse, refractory, or death. RFS was defined as the period from the date of achieving complete remission to the date of relapse or death. The impact of various risk factors on both death and events was assessed through univariate Cox regression. A deliberate selection process was employed to choose variables for reevaluation using a multivariate Cox regression model. The gray test was utilized to estimate and compare the cumulative incidence of relapse (CIR).

Measurable residual disease (MRD) by MFC is a strong and independent prognostic marker of relapse in pediatric AML, yet they are still not sensitive enough, since relapse still occurs in a minority of MRD-negative patients and the opposite is true, not all patients with MRD-positive will relapse (12). Therefore, searching for more sensitive molecular markers is of great importance and molecular evidence of residual leukemia cells can predict relapse several months before clinical emergence (13, 14).

The incidence of WT1 gene overexpression in pediatric AML, as reported in previous studies, ranges from 76% to 83% (9, 15–17), which aligns with the current results. However, Juul-Dal et al. showed a much lower incidence of WT1 overexpression (45%) (1). Previous pediatric studies showed that WT1 gene overexpression was significantly associated with M4 FAB subtype, this goes in agreement with our results (91.4% of M4 cases had high WT1 levels, p=0.018) (9, 18, 19). A high incidence of WT1 overexpression in CBF AML was reported, ranging from 87% up to 100% (p<0.001), while a strong inverse correlation with the presence of KMT2A gene rearrangement (p<0.001) suggesting downregulation of WT1 activating pathway in this leukemia subset, as demonstrated in the present study (1, 9, 20).

The ELN in 2021 recommended WT1 gene expression as a marker for MRD assessment especially in patients without identified markers to follow (2, 7). The current results suggested that failure to achieve 2 log reduction by WT1 MRD post induction I predict relapse (2-year CIR was 33.2% vs 7.9%, p=0.008), but this wasn’t reflected on survival analysis which may be explained by treatment-related mortalities during induction. Previous data published by Lapillonne et al. demonstrated that WT1 levels higher than 50 × 10⁴ ABL copies after induction was an independent prognostic risk factor of relapse (p = 0.002) and death (p = 0.02) in pediatric AML (9). While Shimada et al. reported that high WT1 expression after 1^st induction chemotherapy would be associated with poor outcomes in pediatric AML patients, (5-year OS for WT1 ^+ve was 54.5% vs 79.4% for WT1 ^-ve patients, p=0.036), but multivariate analyses did not confirm it as an independent poor prognostic factor on the outcome of AML patients (OS, p=0.87 and EFS, p=0.92) (21).

In this study, poor responders (WT1 MRD < 2log reduction) post intensification I exhibited notably reduced OS and EFS compared to those with WT1MRD^-ve mostly attributed to a higher cumulative incidence of relapse, which retained its significance as a predictive factor for inferior survival even after accounting for other variables, including initial risk stratification.

These findings align with Cilloni et al. who reported that patients who failed to achieve 2 log reduction by WT1 MRD after intensification therapy were associated with a significant increase in relapse risk (67% vs 42% at 5 years, p= 0.004) (8). Additionally, Nomdedéu et al. analyzed 584 patients under the CETLAM protocol, categorizing them into three groups based on post-intensification WT1 levels (<10, 10.1 to 100, and >100/10⁴ ABL), the OS and LFS were significantly lower among patients with WT1 >100 copies (OS 30%, LFS 24% and CIR 25%, p<0.001) (22).

Furthermore, Candoni et al. reported the impact of MRD negativity before allo-SCT (post intensification I) on the outcome and found that patients with WT1 MRD^+ve (WT1 > 250 copies/10⁴ ABL) had significantly lower OS (HR: 3.9, p<0.0001), DFS (HR: 3.73, p<0.0001) and higher cumulative incidence of relapse (HR 5.06, p< 0.0001) compared to MRD negative patients (23).

One of the main objectives of our study was to pinpoint a subset of patients, especially within the intermediate-risk (IR) group who are at a higher risk of relapse and poorer outcomes. Additionally, we aimed to investigate the concordance between MRD results by MFC and WT1MRD to set the stage for different approaches to upfront therapy for this group. We observed that patients with positive MRD by MFC (≥ 0.1%) after induction I but achieved favorable response by WT1MRD had a significantly lower incidence of relapse. Similarly, Marani et al. demonstrated that flow MRD post-induction did not predict DFS. Approximately 40% of flow-MRD^+ve patients-maintained CR (p=0.41). In contrast, the response by WT1 MRD response was predictive (DFS 46% for WT1MRD^-ve vs 0% for WT1MRD ^+ve, p<0.001) (24). After intensification I, failure to reduce WT1 transcript level identifies a group of patients at significantly increased risk of relapse despite having negative MRD by MFC (RR 60% vs 9.8% with p<0.001; (RFS 38.9% vs 87.5%, p<0.001).

When comparing WT1 as an MRD marker with other monitoring techniques such as PCR for fusion gene transcripts (RUNX1::RUNX1T1, and CBFB::MYH11), similar sensitivities were observed in predicting the relapse among CBF pediatric patients with AML and failure to achieve either 3 log by PCR or 2 log by WT1 post intensification I carry a higher incidence of relapse regardless response by MFC. Similarly, ELN 2021 stated the importance of molecular monitoring of MRD in CBF patients especially at this time point (2).

The current study demonstrates that combined assessment of MRD by WT1 and MFC ably detected patients at a higher risk of relapse (PPV 100%) and identified patients with low relapse risk (NPV 85.7%) as WT1MRD had higher sensitivity while MFC had better specificity among intermediate risk group even with the small number of patients. The same was reported by Guolo et al, where patients with WT1 MRD positive after consolidation therapy had the highest risk of relapse (negative predictive value (NPV): 79%; positive predictive value (PPV): 62%) whereas patients with undetected MFC MRD had a very low relapse risk (NPV: 92%, PPV: 30%) and combination allowed patients to benefit from both the high PPV of WT1 MRD and the high NPV of MFC MRD (25).

Carlo Marani et al. concluded that integrating cytogenetic risk at diagnosis with MRD assessment using both flow cytometry and WT1 allowed for better classification of AML patients: into three prognostic groups: good (no-high risk features “HR” and flow-MRD^-ve), intermediate (no-HR, flow-MRD^+ve and WT1 ≥2 log) and adverse prognosis (HR or WT1 < 2 log) with a 3-year DFS of 78.8%, 51.6% and 0%, respectively, p<.001) (24).

In Conclusion, monitoring measurable residual disease by WT1 expression level either alone (especially in pediatric AML patients without other biological markers) or in combination with other MRD markers may improve the reliability of MRD-based prognostic stratification and thus enable the tailoring of treatment intensity. In addition, suboptimal molecular MRD response (defined as <3 log reduction by qPCR or <2 log reduction by WT1) in AML pediatric patients with CBF may influence frontline therapy decisions due to the increased risk of relapse. Therefore, all study groups now apply extensive biological characterization of the AML cells and response based on MRD assessments by both MFC and molecular technique for better risk-group adapted treatment (26).

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author.

The studies involving humans were approved by Children’s Cancer Hospital Egypt Scientific Advisory Committee (SMAC) and the Institutional Review Board (IRB). The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent for participation in this study was provided by the participants’ legal guardians.

SA: Conceptualization, Writing – original draft. ME: Conceptualization, Data curation, Writing – original draft. DY: Methodology, Validation, Writing – original draft. NE: Methodology, Writing – original draft. AM: Methodology, Writing – original draft. NY: Formal analysis, Writing – original draft. AmE: Formal analysis, Writing – original draft. HH: Supervision, Writing – review & editing. EK: Writing – review & editing. AlE: Supervision, Writing – review & editing.