Prevalence of SNP rs16754
All available specimens (n = 790) were subjected to direct sequencing of WT1 exon 7 to assess WT1 SNP status. SNP rs16754 represents an A>G substitution at nucleotide position 1293, the third position of codon 352 encoding arginine (CGA>CGG). At least 1 copy of the minor allele was detected in 229 patients (29.0%), 38 (16.6%) of whom were homozygous for the SNP (191 were heterozygous).
Characteristics of the Study Population
Demographic, laboratory, and clinical characteristics of patients with and without WT1 SNP rs16754 were compared (). There were no significant differences in sex, age, median diagnostic blast percentage, median diagnostic WBC count, or French-American-British class between SNP-positive and SNP-negative patients. There were significant differences in the racial distribution of the minor SNP allele. SNP rs16754 occurred at the highest frequency in patients of Asian (66%) and Hispanic (42%) descent and was less frequent in white (24%) and African American (22%) patients. Patients with the SNP had a lower prevalence of inversion 16 (6.3% v 12.6%; P = .043), and higher prevalence of −5/del(5q), (3.1% v 0.8% P = .047) compared with their SNP-negative counterparts. No other association with cytogenetic groups were identified.
Characteristics of Patients With and Without WT1SNP rs16754
The association between SNP rs16754 and other molecular alterations was investigated. FLT3/ITD, NPM1, and CEBPA mutations occurred at comparable frequencies in patients with and without the WT1 SNP (). Compared with the SNP-negative patients, who had a WT1 mutation prevalence of 10.4%, WT1 mutations were identified in only 3.2% of those with the SNP (P = .002). Further evaluation demonstrated that none of the 38 patients homozygous for SNP rs16754 had a concomitant WT1 mutation ().
Prevalence of Wilms' tumor 1 (WT1) mutation in patients with homozygous and heterozygous single nucleotide polymorphism (SNP) rs16754 compared with WT1 wild-type patients.
The proportion of SNP-positive and SNP-negative patients was similar when they were stratified into high-risk (−7, −5/del(5q), FLT3/ITD with high allelic ratio), low-risk (t,(8,21) inv(16)/t,(16,16) NPM1, or CEBPA mutations) or standard-risk groups (all other patients). Minimal residual disease data from the end of course 1, assessed by multidimensional flow cytometry, were available from 184 patients enrolled on AAML-03P1. Minimal residual disease higher than 0.01% was detected in 13 (24%) of 55 SNP-positive patients, compared with 40 (31%) of 129 SNP-negative patients (P = .405).
Clinical Outcome and Prognostic Impact of WT1 SNP
Clinical outcome data were examined for the patients with known WT1 SNP rs16754 status (). Patients with or without SNP rs16754 had similar CR rates after one course of induction (81.2% v 80.2%, P = .837). Overall survival at 5 years from study entry for SNP-positive patients was 60% (SE [±] 7%) versus 50% ± 5% for those without the SNP (HR, 0.76; P = .031). The corresponding DFS from CR was 51% ± 8% and 47% ± 5% for those with and without SNP rs16754 (HR, 0.86; P = .415). For patients who achieved an initial remission (n = 615), those with and without the SNP had similar relapse rates (40% ± 8% v 43% ± 5%; HR, 0.92; P = .763) and similar treatment-related mortality (8% ± 4% v 11% ± 3%; HR, 0.79; P = .442).
Fig 2. Prognostic significance of Wilms' tumor 1 single nucleotide polymorphism (SNP) rs16754 mutations in pediatric acute myeloid leukemia. Clinical outcome for patients with and without SNP rs16754. Kaplan-Meier estimates of (A) overall survival and (B) disease-free (more ...)
We performed univariate and multivariate Cox regression analysis to evaluate whether presence of the WT1 SNP as well as other known prognostic factors (ie, cytogenetics, mutations, diagnostic WBC, and race) were predictors of OS and RFS in the entire study cohort (). Patients with SNP rs16754 had an improved survival with an HR of 0.76 for death from enrollment (P = .031), and an HR of 0.92 for relapse from achieving an initial remission (P = .6). Patients with high-risk features had an HR of 1.76 for death from diagnosis (P < .001) and an HR of 1.91 for increased risk of relapse (P < .001), whereas low-risk group assignment was associated with improved survival (HR, 0.48; P < .001) and lower risk of relapse (HR, 0.49; P < .001). Diagnostic WBC higher than 50 ×109/L was a predictor of decreased OS (HR, 1.30; P = .005) and higher risk of relapse (HR, 1.26; P = .047). Non-white patients had decreased OS (HR, 1.34; P = .001) but not significantly reduced RFS (HR, 1.17; P = .152).
Univariate and Multivariate Cox Regression Analysis of WT1SNP rs16754 and Other Validated Risk Factors
In the multivariate analysis, WT1 SNP rs16754 was an independent prognostic marker for improved OS with an HR for death of 0.64 compared with SNP-negative patients (P = .004). In this model, HR for relapse from remission for patients with WT1 SNP was 0.79 compared with their SNP negative counterparts (P = .197).
Prognostic Significance of SNP rs16754 in Risk Groups
The prognostic impact of SNP rs16754 was evaluated in specific clinical risk groups (). In patients with standard-risk disease (no high or low risk features, n = 294), WT1 SNP was identified in 79 patients (26%). Those with and without SNP rs16754 had identical OS at 5 years from diagnosis of 47% and a similar RR (48% ± 14% v 45% ± 9%; P = .77) and DFS (45% ± 14% v 47% ± 9%; P = .88) from remission. WT1 SNP was identified in 58 patients (26%) with low-risk AML (CBF translocations, CEBPA or NPM1 mutations, n = 221). In contrast to standard-risk patients, low-risk patients with SNP rs16754 had an actuarial OS of 90% ± 8%, versus 64% ± 9% for low-risk SNP-negative patients (HR, 0.27; P = .001). In low-risk patients who achieved an initial remission, DFS at 5 years from remission for SNP-positive patients was 72% ± 13% versus 53% ± 10% for SNP-negative patients (P = .041), with a corresponding RR of 21% ± 12% versus 41% ± 9% (HR, 0.49; P = .179) compared with their SNP-negative low-risk counterparts.
Fig 3. Prognostic impact of Wilms' tumor 1 single nucleotide polymorphism (SNP) rs16754 in specific clinical risk groups. Estimates of the probability of (A, D, G) overall survival, (B, E, H) disease-free survival, and relapse risk (C, F, I) for (A, B, C) low-risk, (more ...)
In patients with high-risk disease (−7, −5/del5q or high risk FLT3/ITD, n = 94), the WT1 SNP was identified in 33 patients (35%). Actuarial OS at 5 years from diagnosis was 41% ± 22% for SNP-positive patients versus 21% ± 12% for SNP-negative patients (P = .083). In high-risk patients who achieved an initial remission, DFS at 5 years from remission for SNP-positive patients was 30% ± 22% versus 10% ± 11% for SNP-negative patients (P = .273), with a corresponding RR of 60% ± 23% versus 78% ± 15% (P = .179) compared with their SNP-negative counterparts.
We further evaluated the prognostic significance of SNP rs16754 in patients with normal karyotype. Of the 125 patients without cytogenetic abnormalities, 38 had SNP rs16754 (30%). Overall survival at 5 years from study entry for patients with and without WT1 SNP was 45% ± 18% versus 39% ± 12% (P = .522). In patients who achieved an initial remission, DFS at 5 years from remission for SNP-positive patients was 51% ± 20% versus 41% ± 14% for SNP-negative patients (P = .273), with a corresponding RR of 46% ± 20% versus 47% ± 14% (P = .882) compared with their SNP-negative counterparts.
Within the SNP-positive group, homozygous (n = 38) and heterozygous (n = 191) patients had similar 5-year OS (61% ± 8% v 56% ± 18%, P = .960), DFS (46% ± 8% v 41% ± 17%, P = .720), and RR (38% ± 9% v 50% ± 20%, P = .312).
SNP rs16754 Is Present in the mRNA Transcript
Because the presence of this synonymous SNP imparts prognostic significance, we sought to determine a potential mechanism by which a silent polymorphism may be translated into a functional alteration. Differential RNA editing is one mechanism by which a silent genomic mutation may create a functional alteration in the pre-mRNA transcript, leading to a change in the protein product. An example of RNA editing has been previously described in codon 280 of WT1
To determine whether differential RNA editing might occur as a result of the synonymous SNP rs16754, we sequenced WT1
exon 7 from cDNA transcripts obtained from 50 patients at random from COG AAML-03P1. Of the 50 patients, 13 (26%) had at least 1 minor allele of the WT1
SNP; 4/13 SNP-positive patients were homozygous for the SNP. Correlation of genotyping data performed on genomic DNA with these cDNA data showed identical sequences, demonstrating that RNA editing is not involved in the pathogenesis of AML in regard to SNP rs16754.
SNP rs16754 Is Associated With Elevated mRNA Expression
is highly expressed in most patients with AML.13
We questioned whether patients with SNP rs16754 may have an altered WT1
expression level. WT1
expression levels were evaluated by quantitative reverse-transcriptase PCR in 114 unselected patient samples from COG-AAML03P1 with known WT1
SNP rs16754 (SNP positive, n = 30) and WT1
mutation (mutation positive, n = 13) status (). WT1
expression levels for each sample were normalized to the expression in normal marrow control. WT1
expression was highly variable, ranging from 0.00- to 2949.42-fold normal marrow expression (median 70.4-fold normal bone marrow). Eleven patients did not have detectable WT1 expression (10%) and 97 patients (85%) had WT1 expression higher than observed in normal marrow controls.
Levels of Wilms' tumor 1 (WT1) expression by single nucleotide polymorphism (SNP) and mutation status (Mut). Median expression within each cohort is indicated by the dashed line. NBM, normal bone marrow.
Median WT1 expression in WT1 wild-type patients (without SNP rs16754 or a WT1 mutation, n = 71) was 40.61 times that of normal marrow controls (range, 0.00 to 2949.42), whereas the median expression in patients with the WT1 SNP (n = 30) was 215.76 times that of normal marrow control (range, 0.00 to 1,399.23; P = .0214). We further compared WT1 expression levels in those with and without WT1 mutations. Of the 114 patient samples tested, 13 (11.4%) were positive for a WT1 mutations (12 exon 7 mutations, one exon 8 mutation) and only one patient harbored both SNP rs16754 and a WT1 mutation. Median WT1 expression level for this cohort was 327.14-fold normal marrow control (range, 54.17- to 902.3-fold; P = .0005) compared with the WT1 wild-type cohort.