Wilms’ Tumor Gene (WT1) Expression in Cytogenetically Distinct Subsets of AML
Sanjive Qazi11-5 and Fatih Uckun2-5*
1Bioinformatics Program, Gustavus Adolphus College, St. Peter, MN 56082, USA
2AresMIT Biomedical Computational Strategies (ABCS), Minneapolis, MN 55402, USA
3Ares Pharmaceuticals, LLC, St. Paul, MN 55110, USA
4Division of Hematology-Oncology, Department of Pediatrics USC KSOM, LA, CA 90027, USA
5Norris Comprehensive Cancer Center, University of Southern California Keck School of Medicine (USC KSOM), Los Angeles, CA 90027, USA
*Corresponding author: Fatih Uckun, AresMIT Biomedical Computational Strategies (ABCS), Minneapolis, MN 55402, USA, Tel: +1 6128106032; E-mail: firstname.lastname@example.org
Citation: Qazil S, Uckun F (2018) Wilms’ Tumor Gene (WT1) Expression in Cytogenetically Distinct Subsets of AML. J Blood Transfus Hematop 2018: 1-4. doi:https://doi.org/10.29199/BTHP.101014
Received: 02 January, 2018; Accepted: 24 January, 2018; Published: 10 February, 2018
The Wilms’ Tumor Gene (WT1) [1-3] is a tumor-suppressor gene associated with Wilms’ tumor, a pediatric kidney cancer. The protein product of the WT1 gene is a zinc-finger transcription factor and a tumor-associated antigen. While WT1 is an intracellular protein, WT1-derived peptides are expressed on the surface of cancer cells in the context of HLA Class I antigens. As such they can be targeted by Bispecific T-cell Engagers (BiTEs), CAR-T cells and WT1-specific CTL that expand after WT1 peptide vaccine treatments [4-19].
WT1 is abundantly expressed in numerous hematological malignancies, including Acute Myeloid Leukemia (AML) [3,20-25]. WT1 expression may be induced by leukemia-associated oncogenes (like PML-RARA) as a physiologic, inhibitory response to oncogenic stress. In this case, subsequent inactivating mutations in WT1 would provide an additional growth advantage . Oncomine database survey of WT1 mRNA expression in AML vs. non-leukemic healthy bone marrow samples showed 2-23-fold higher expression levels in AML samples with highly significant P-values for all studies deposited in the Oncomine database (Table 1) [26-29].
Table 1: Oncomine Survey of WT1 mRNA Expression in AML vs Healthy Non-Leukemic Bone marrow samples.
We utilized an online data mining tool harboring 715 datasets with 86733 samples for cancer patients and cell lines to assess expression of WT1 across major cancer subtypes (https://www.oncomine.org/resource/main.html). We focused our analysis on mRNA expression comparing non-leukemic normal versus AML samples (5 independent comparisons, log2 median centered expression values for each data set) and filtered expression signals that were greater than 1.5-fold difference and p-value from two sample t-tests less than 0.001. WT1 expression was ranked according to Fold change. The table depicts the T-statistic, P-value, sample sizes for non-leukemic normal and AML samples for each study that were also referenced to the Gene Expression Omnibus identifier (GSE#). All 5 studies [26-29] in the Oncomine database exhibited significant increase in WT1 expression for AML samples (Fold change ranged from 2 to 23 relative to non-leukemic bone marrow samples).
We next interrogated the GSEA13159 database of gene expression data from the MILE Study (Microarray Innovations in Leukemia), that were generated utilizing the Affymetrix Human Genome U133 Plus 2.0 Array platform, for WT1 mRNA expression in cytogenetically distinct subsets of AML patients (N=542). The MILE study compared gene expression profiles for subsets of leukemia defined by gold standard laboratory techniques, and controlled for statistical batch effects from hybridizations measured at different clinics utilizing pre-processing methods including DQN3 normalization of the detected signals.
Figure 1: Increased levels of WT1 expression in AML subsets compared to normal samples from the MILE study. Gene expression profiling data utilizing the Affymetrix Human Genome U133 Plus 2.0 Array from the MILE Study were interrogated for WT1 expression in AML samples relative to normal non-leukemic healthy bone marrow samples. DQN3-normalized expression levels for the WT1 probeset, 206067_s_at in cytogenetically distinct AML subsets, including AML with t(8;21)(q22;q22.1) translocation, AML with inversion (inv)(16)(p13.1q22) or t(16;16)(p13.1;q22) translocation, AML with t(15;17)(q24.1;q21.2) translocation (APL with PML-RARA according to WHO classification), AML with 11q23 rearrangements involving the MLL gene (t(11q23)/MLL-R) including t(9;11)(p21.3;q23.3), AML with a complex aberrant karyotype, and AML with a normal karyotype or other abnormalities (e.g., t(1;22)(p13.3;q13.3), inv(3)(q21.3q26.2) or t(3;3)(q21.3;q26.2), t(6;9)(p23;q34.1), are depicted using mean and standard error values presented in the bar chart. AML with inv (16) and AML with t(15;17) exhibited the highest expression levels for WT1. MLL-R, MLL gene rearranged. t, translocation. inv, inversion. BM, bone marrow.
As detailed in Table 1, AML samples exhibited 4.9-9.7 fold higher expression levels of WT1 than normal bone marrow samples and the documented differences were highly statistically significant. Further, several of the WT1-regulated genes, such as EWSR1, WTAP, U2AF2, and PAWR were also expressed at significantly higher levels in AML samples . As depicted in Figure 1 and Table 2, samples from AML patients with inv(16) (9.3-fold amplification, P<10-16) or t(15;17) (9.7-fold amplification, P<10-16) showed the highest WT1 expression levels.
Table 2: WT1 expression in cytogenetically distinct AML subtypes (N=542).
One way ANOVA was performed across cytogenetically distinct subsets of AML in the MILE study and the statistical significance for the observed differences (vs. non-leukemic normal control samples) was assessed using the Dunnett's post hoc tests. Mean Fold change and P-values following the Dunnet’s post hoc tests are depicted in the table. Compared to normal samples, leukemic samples from each of the 6 AML subsets exhibited greater than 5-fold increase in WT1 expression levels for the WT1 probeset, 206067_s_at. CON: normal control.
In AML, the levels of WT1 gene expression correlate with prognosis: Patients with high-level expression have a lower likelihood to respond to induction chemotherapy and achieve complete remission . A short 4-gene signature associated with high WT1 expression levels and the resultant 4-gene expression score were found to be predictive of adverse prognosis in AML . A recent meta-analysis confirmed the significantly poor prognostic impact on OS and DFS of patients with AML in total population and some specific subgroups . Therefore, the level of WT1 expression can add prognostic information in AML risk stratification . High WT1 expression has been shown to be an early predictor of relapse in patients with acute promyelocytic leukemia in remission . Candoni et al., analyzed the outcome of allogeneic hematopoietic stem cell transplantation in a population of FLT3-positive AML patients according to molecular Minimal Residual Disease (MRD) at the pretransplant workup, assessed by the quantitative expression evaluation of the WT1 gene . Patients with undetectable WT1 mRNA levels in their pre-transplant remission bone marrow (i.e., a WT1-negative complete remission) before had a very good outcome with low relapse rate post-transplant; conversely, patients whose bone marrows tested positive for WT1 mRNA (i.e., WT1-positive complete remission) consistent with the presence of a molecular MRD as well as refractory/relapsed patients had a poor outcome . The authors concluded that the WT1 MRD stratification in FLT3-positive AML may serve as a valuable tool to identify patients who are at a high risk for relapse and might benefit from post-transplant intervention with FLT3 inhibitors . Dulery et al., reported the usefulness of peripheral blood MRDWT1 monitoring in identifying very high-risk patients, who could benefit from an early preemptive treatment, and those who do not need such an intervention .
Several WT1-specific targeted cancer immunotherapy modalities, including therapeutic vaccines, WT1-specific CTL or CAR-T cell platforms, are currently being evaluated in clinical trials for multiple therapeutic indications, including AML [4-19]. Scheinberg et al., have been pursuing WT1 peptide vaccines (viz., galinpepimut-S) and recently reported novel synthetic WT1 peptide designs for more effective immunization/CTL generation using advanced computer modeling tools . Further, Advaxis and Sellas (a subsidiary of Galena Biopharma - (NASDAQ:GALE) completed a licensing agreement for using a WT1-targeted heteroclitic peptide antigen mixture (galinpepimut-S/GPS licensed from Memorial Sloan Kettering Cancer Center) in the context of the Advaxis’ Lm-based antigen delivery technology aimed at reducing immune tolerance in the tumor microenvironment. SELLAS has been granted orphan drug designation from the FDA and the European Medicines Agency (EMA) and been given FDA fast track status.
Recently, a T-Cell Receptor (TCR) that specifically reacts with the WT1 peptide in the context of HLA-A*24:02 has been identified. This WT1-specific TCR-gene was transduced to autologous T-cells using a retroviral vector encoding small interfering RNAs for endogenous TCR genes to generate WT1-specific TCR-T cells capable of executing an immune reaction to WT1-expressing AML cells. The early results of the first-in-human Phase 1 clinical trial (www.umin.ac.jp as #UMIN000011519) of this WT1 TCR transduced T-cell therapy (also known as WT1 antigen-specific RetroNectin TCR gene therapy - Takara Bio) in therapy-refractory AML patients have been reported . Likewise, ATA520 (Atara Biotherapeutics) is a Cytotoxic T Lymphocyte (CTL) product candidate against WT1 (i.e., WT1-CTL) that is being studied in multiple clinical trials for its safety and activity as a targeted biotherapeutic agent against WT1-positive hematologic malignancies. Shah et al., reported the safety and feasibility of a WT1 peptide-loaded donor-derived Dendritic Cell (DC) vaccine given with donor lymphocyte infusions designed to enhance and direct the graft-versus-leukemia effect . Preliminary results indicate the DC-based vaccination is safe and feasible after allogeneic HCT, and suggest that this strategy could potentially be used to sensitize the repopulated allogeneic-donor immune system to WT1+ leukemia cells.
Current Challenges and Future Directions
The landscape of available treatment strategies for AML has changed dramatically with the recent FDA approval several targeted therapeutic agents with clinical activity in AML . Recent vaccination approaches to target WT1 have been hampered by poor in vivo immune potency, likely due to the conserved self-antigen nature of WT1. That is why some groups are exploring the use of synthetic consensus DNA vaccines for breaking tolerance to this germline antigen . Even in AML with very high level WT1 expression, the T-cell responses and prolonged remissions reported in AML patients treated with this approach have generally been limited to the setting of MRD (i.e., not observed in the setting of overt disease). Engineered CAR-T cells or WT1-CTLs are more likely to overcome these inherent challenges of poor immunogenicity of WT1.