Standardization of WT1 mRNA Quantification for Minimal Residual Disease (MRD) Monitoring in Acute Leukemia Patients: A European LeukemiaNet Concerted Action.

Detection of minimal residual disease (MRD) has proven to provide independent prognostic information for treatment stratification in several types of leukemia. In acute myeloid leukemia (AML), the reliability of real-time quantitative PCR (RQ-PCR) and its potential clinical value for MRD studies using fusion gene (FG) transcripts as PCR targets such as PML-RARa, AML1-ETO and CBFb- MYH11 has been demonstrated, but these markers are present only in a minority of cases. In order to overcome this problem several groups looked for alternative markers and growing evidence has suggested that the high expression of WT1 in a significant proportion of acute leukemia cases provides a suitable target for therapy as well as for monitoring of MRD. However, heterogeneity of molecular approaches resulted in a lack of comparability between different MRD studies. This has been solved using RQ-PCR in a network of 9 laboratories within the European LeukemiaNet. Overall 8 primer/probe sets were evaluated including published and “in-house” sets. The assays analyzed differed significantly in terms of efficiency and sensitivity. Three assays with superior performance were identified, achieving sensitivities of at least 1 in 10,000 in serial dilutions of HL60 cells (WT1 positive). Subsequent analysis of two of the primer/probe sets, which amplify ex. 6/7 and 7/8 of WT1 respectively, revealed the potential for false negative results, following documentation of deletions of the WT1 gene in this region in primary AML samples. In two of these cases, different deletions of sequences corresponding to part of WT1 ex.8 were documented by WT1 RNA sequencing. Therefore, a primer/probe set amplifying ex. 1/2 of WT1 has been subject to further analysis in a QC round involving 9 labs. The analysis of 33 normal BM, 32 normal PB and 12 CD34 enriched PBMNCs gave the following results: BM, mean 70,32 WT1 copies/10⁴ ABL copies (range 8,99–209,82); PB, mean 3,30 WT1 copies/10⁴ ABL copies (range 0–13,55); CD34 enriched PBMNCs, mean 8,16 WT1 copies/10⁴ ABL copies (range 1,55–26,16). Overall, these analyses underline the importance of standardization in the development of RQ-PCR assays for MRD detection in leukemia. There is increasing interest in identification of genes that are over-expressed in leukemia as potential MRD targets. However, it is clear that incorporation of such MRD targets into risk-directed treatment protocols is critically dependent upon establishing thresholds of expression in normal blood and marrow on regeneration following myeloablative therapy.