ERG, BAALC, and MN1 RNA Levels Are Correlatively Over-Expressed, and Relate to Risk-Defining Prognostic Mutations in Intermediate-Risk Acute Myeloid Leukemia

A variety of prognostic molecular markers have been proposed for risk stratification of intermediate-risk AML, which includes patients with normal cytogenetics (CN) and patients with cytogenetic abnormalities other than the recurrent prognostically informative cytogenetic aberrations (CA). Acquired mutations of FLT3 ITD, NPM1, and CEBPA have well-established prognostic significance, and have been incorporated into consensus classification schemes. In addition, several gene expression markers (WT1, ERG, BAALC, and MN1) have been proposed as prognostic indicators, but with less extensive validation. Whether these new gene expression markers should be incorporated into proposed “molecular risk scores” for AML has not been well refined. In this study, we have determined the transcript levels of WT1, ERG, BAALC, and MN1 in AML patients, and correlated these quantitative levels with other validated clinical and laboratory prognostic indicators. Methods: Diagnostic blood or bone marrow samples were analyzed from 56 pre-treatment AML patients from our institution (48% male; median age 59). Metaphase cytogenetics showed 4 patients with inv(16), 4 with a monosomal karyotype, 25 with a CN karyotype and 23 with a CA karyotype (intermediate-risk cytogenetics). The expression levels of WT1, ERG, BAALC, and MN1 were quantified with TaqMan real-time PCR and normalized to ABL. The FLT3 ITD, NPM1, DNMT3A R882, CEBPA, and MLL PTD mutations were detected with either HRM, direct sequencing, and/or PCR fragment analysis. The WT1 SNP rs16754 was determined with a TaqMan genotyping assay. Results: In the 56 AML patients, the prevalence of the FLT3 ITD, NPM1, MLL-PTD, and DNMT3A R882 mutations were similar to previously reported values at 37%, 27%, 6%, and 13%, respectively. The frequency of patients carrying the minor allele of the WT1 SNP (combined heterozygous and homozygous) was 30%. Compared with their expression level in 29 normal control individuals, all four genes were significantly over-expressed in the AML patients, with mean RNA level differences of 1.98-log for WT1 (P<0.0001), 1.18-log for ERG (P<0.0001), 1.02-log for BAALC (P<0.0001), and 0.53-log for MN1 (P = 0.005). ERG, BAALC, and MN1 were expressed at low but detectable levels in 100% of the normal control samples, while WT1 was not detectably expressed in 48% of the controls. For all 4 genes, there were no significant differences in the expression levels between the CA & CN cytogenetic subclasses. As high transcript levels of WT1, ERG, BAALC, and MN1 have been proposed to impart a high risk AML phenotype, one might predict that the RNA levels of each of these 4 genes would correlate with one another. To confirm that hypothesis, we found a highly significant correlation of quantitative RNA levels among the ERG, BAALC, and MN1 genes (P <.004; R=0.42–0.72). WT1 RNA levels were highly correlated with ERG (P<0.0001; R=0.59), but less highly correlated with BAALC & MN1 (P=0.04; R=0.3). Categorization of the gene expression level as “high” or “low” (above or below the median) also revealed a similar relationship, in which ERG, BAALC, and MN1 RNA were all significantly correlated (P<0.01), but there was no significant correlation of WT1 RNA with the other 3 genes (P>0.9). Consistent with its role as a prognostic risk marker, ERG transcript levels were significantly lower in CN-AML patients with the low-risk NPM+/FLT3- genotype than in those with other genotypes (0.42 log expression difference; P<0.04). Consistent with WT1's role as a risk marker, in the CA-AML patients, WT1 RNA levels were significantly higher in patients with the high-risk FLT3+ genotype than in those with other genotypes (1.3 log expression difference; P<0.04). Conclusions: To better define prognostic risk markers in AML, we have performed comprehensive mutation and gene expression analysis on 56 diagnostic AML samples. WT1, ERG, BAALC, and MN1 RNA were significantly over-expressed in AML patients compared to normal controls. ERG, BAALC, and MN1 RNA levels significantly correlated with one another. The prevalence of FLT3, NPM, DNMT3A, and MLL mutations in these AML patients was predictably high, and some of these well-characterized prognostic mutations were significantly associated with gene expression levels. Additional details on the relationship of these new prognostic RNA markers to the clinical outcome of this AML cohort will be reported at the meeting.

No relevant conflicts of interest to declare.