Priya Virappane

PURPOSE: To determine the clinical relevance of Wilms' tumor 1 (WT1) gene mutations in acute myeloid leukemia (AML) with normal karyotype (NK). PATIENTS AND METHODS: Exons 7 and 9 of WT1 were screened in samples from 470 young adult NK AMLs using a combination of direct sequencing and high-resolution capillary electrophoresis. RESULTS: Overall, 51 mutations were detected in 47 cases (10%): 46 frameshift mutations with insertion/deletion of one to 28 base pairs in exon 7 (n = 45) or exon 9 (n = 1), with a median mutant level of 45% (range, 8% to 86%), and five substitutions in exon 9: D396N (n = 3), H397Y (n = 1) and H397Q (n = 1). Patients with WT1 mutations had an inferior response to induction chemotherapy compared with wild-type cases (complete remission rate, 79% v 90%, odds ratio [OR] = 3.02; 95% CI, 1.17 to 7.82; P = .02), a higher rate of resistant disease (15% v 4%; OR = 9.33; 95% CI, 2.38 to 36.6; P = .001), an increased cumulative incidence of relapse (67% v 43%, hazard ratio [HR] = 3.02; 95% CI, 1.69 to 5.38; P = .0008), with a reduction in both relapse-free survival (22% v 44%; HR = 2.16; 95% CI, 1.32 to 3.55; P = .005) and overall survival (26% v 47%; HR = 1.91; 95% CI, 1.23 to 2.95; P = .007) at 5 years. In multivariate analysis, which included FLT3 internal tandem duplication and NPM1 mutation status, the presence of a WT1 mutation remained an independent adverse prognostic factor. CONCLUSION: WT1 mutations are a negative prognostic indicator in NK AML and may be suitable for the development of targeted therapy.

WTX (Wilms’ tumor gene on the X chromosome) is inactivated in 30% of Wilms’ tumors, mostly by chromosomal deletion.[1][1] The WTX protein is a component of the β-catenin destruction complex and promotes its ubiquitination and degradation, functioning as a negative regulator of WNT/β-catenin

Abstract The Wilms’ tumour 1 (WT1) gene is highly expressed in various types of leukemia, suggesting a role both as a target for immunotherapy and a means for monitoring minimal residual disease. More recently WT1 has been recognised as an important mutational target in normal karyotype (NK) acute myeloid leukemia (AML), with clustering of mutations to exons 7 and 9. In this study, we set out to determine the clinical relevance of these mutations. Diagnostic DNA samples were obtained from peripheral blood or bone marrow of 470 young adult AML patients entered into the UK Medical Research Council AML 10 and 12 trials. Mutation analysis was performed using standard PCR-based direct sequencing and/or high-resolution capillary electrophoresis of exons 7 and 9. Overall, 51 mutations were observed in 47 cases (10%). Of these, 42 cases had 46 frameshift mutations arising from insertions (n=41) or deletions (n=5) of between 1 and 28 bps that would result in disruption of the C-terminal DNA binding domain of the protein. Median mutant level was 45% (range 8–86%). The remaining 5 cases were non-synonymous amino acid substitutions in exon 9, D396N (n=3) which is implicated in Denys-Drash syndrome, and 1 each of H397Y and H397Q that are of unknown functional significance. There was a borderline inverse relationship between the presence of WT1 mutations and NPM1 exon 12 mutations (p=.05), and an increased proportion of FLT3-ITDs in WT1 mutant cases (p=.08). In order to determine the clinical relevance of WT1 mutations we compared patient characteristics between WT1 mutated and WT1 wild-type cases. There was no difference between the 2 groups in age, gender, presenting WBC, or type of AML (de novo/secondary). Patients with WT1 mutations had an inferior response to therapy compared to WT1 wild-type cases (complete remission [CR] 79% Vs 90% respectively, odds ratio [OR] 3.02, 95% confidence intervals [CI] 1.17–7.82, p=.02) which was due to a higher rate of resistant disease (RD) (15% Vs 4%, OR 9.33, CI 2.38–36.6, p=.001). In univariate analysis mutated cases also had a significantly reduced disease free survival (DFS), 22% Vs 44% at 5 years (OR 2.16, CI 1.32–3.55, p=.005) and overall survival (OS), 26% Vs 47%, OR 1.91, CI 1.23–2.95, p=.007). In multivariate analysis considering age, gender, type of leukemia, WBC, FLT3-ITD and NPM1 mutant status, a WT1 mutation remained an independent adverse prognostic factor for both response to therapy (CR: OR 3.19, CI 1.25–8.13, p=.01; RD: OR 4.93, CI 1.58–15.37, p=.006) and survival (DFS: OR 1.60, CI 1.07–2.38, p=.02; OS: OR 1.52, CI 1.05–2.18, p=.03). These results indicate that WT1 mutations are an additional prognostic marker in NK AML and may be suitable for targeted therapy.

Abstract The molecular mechanisms involved in histological transformation of follicular lymphoma (FL) to diffuse large B-cell lymphoma (DLBCL) are largely unknown. Here we investigated the clonal relationship in FL and DLBCL in 17 cases of paired FL/DLBCL samples and also in a bone marrow transplant (BMT) model. The aim of this study was to determine the cell of origin of the transformed DLBCL (t-DLBCL) and whether this cell always arises as a clonal evolution from the major FL clone. The immunoglobulin variable heavy chain (IgVH) was amplified by PCR using family specific primers, the major clone identified by homo/heteroduplex and genealogical trees generated. As expected 70% of FL/DLBCL cases used VH3, 24% VH4 and 6% VH5. The median degree of somatic hypermutation (SHM) was 15% (range 4 to 43%). In 3/17 cases the pattern of SHM was identical between FL and DLBCL. In 6/17 cases a clear pattern of direct evolution was evident. Surprisingly for 8/17 cases the pattern of SHM was compatible with the FL and DLBCL arising from a precursor cell rather than direct evolution. Clone specific primers and probes were designed to investigate whether there was evidence of the t-DLBCL in the earlier biopsy. Quantification by RQ-PCR demonstrated that in most cases the transformed clonotype was already present at levels between 10-4 and 10-2. We further examined an interesting case in which both the father (donor) and the son (recipient) developed FL and t-DLBCL 3 and 10 years after transplantation respectively. Sequencing confirmed the presence of an identical t(14;18) translocation in donor and recipient and that both cases used IGHV3-48*03. Analysis of the genealogical trees of SHM in these cases was also consistent with the evolution of t-DLBCL from a common precursor cell rather than direct evolution. These data are consistent with two distinct patterns of transformation of FL to t-DLBCL. About 50% of our cases had evidence of direct evolution while in the remaining 50% the pattern is more suggestive of the FL and t-DLBCL arising from a common precursor cell. The long latency period after BMT is in agreement with the requirement for additional genetic events within the t(14;18) bearing post germinal centre precursor lymphoma cell for the development of these diseases. The finding of the t-DLBCL clonotype in the FL sample before transformation suggests that the transforming event may occur in a minor subclone of the disease or earlier in a lymphoma precursor cell. Ongoing experiments seek to investigate the genetic events arising in these cells that result in transformation.