WT1 Gene Expression Limited Application for MRD Monitoring in AML Patients Carrying MLL Rearrangements.

WT1 gene expression (GE) is widely used for monitoring of minimal residual disease (MRD) in acute myeloid leukemia (AML) patients. Predictive value of WT1 GE in MLL-positive AML is not clear. Comparison of WT1 GE and fusion genes (FG), detected at RNA and DNA levels is rarely done. Here we present comparison of MRD data, assessed by WT1 GE, FG transcripts and MLL fusions alleles (MFA).

27 samples from 5 MLL-positive AML patients (pts) were analyzed. Median age was 13 years (range 0.8-58 years). 5 samples were obtained at diagnosis, 21 samples during follow-up treatment, 1 at diagnosis of relapse. Initial diagnostics included cytomorphology, immunophenotyping of blast cells, cytogenetics. Presence of MLL rearrangements was detected by FISH, reverse-transcriptase PCR (RT-PCR) and confirmed by long-distance inverse PCR. Among 5 pts there were two MLL-MLLT4-positive cases, two MLL-MLLT10-positive. One patient carried MLL-MLLT3 fusion gene. Quantification of MFA in genomic DNA by patient-specific primers-probe combinations and detection of FG transcript kinetics in RNA/cDNA during treatment were performed by RQ-PCR as previously described (T. Burmeister et al, Leukemia 2006, 20; J. Gabert et al, Leukemia, 2003, 17, respectively). Due to high concordance rate (96%) between qualitative results obtained by FG transcript and MFA detection, these results were combined into one group named as standard methods (SM) of MRD monitoring. Sample was defined as positive if only one result obtained by SM was positive. Sample was defined as negative if both results assessed by FG transcript and MFA were negative. Quantification of WT1 was performed in bone marrow (BM) samples by real-time quantitative PCR (RQ-PCR) with normalization to ABL gene. Additionally, BM samples taken from 14 pts without hematological malignancies were investigated. WT1 threshold level (TL) calculation was based on ROC curve analysis. TL was defined as value with maximal sensitivity and specificity.

ROC analysis of WT1 GE revealed an area under curve (AUC) of 0.890 (95% CI 0.800-0.990). TL was set at 32 with sensitivity of 62% and specificity of 100%. Value of TL was equal to upper level of WT1 normalized copy number (NCN) in BM of 14 pts without hematological malignancies (median 10, range 0-32 WT1 copies/10⁴ABL copies). In AML pts it was detected 13 WT1-positive samples (48%). 21 samples (78%) were positive by SM. 6 samples (22%) were negative both by SM and WT1 GE. Discrepant results were found in 8 samples (30%) (table 1). These discrepant results were observed in follow-up samples only. Qualitative concordance between SM and WT1 GE was 70% in our series.

Based on the ROC curves analysis it was not found particular TL of WT1 NCN that clearly distinguishes MRD-negative pts from those with positive MRD, detected by SM. Applicability of WT1 NCN quantification for MRD monitoring in the assessed MLL-rearranged AML samples appears to be limited because of relatively high percentage of false-negative samples. Discrepant results could be explained by high variability of WT1 GE during antileukemic therapy, and presence of WT1 GE in normal BM. Thus WT1-negative results should be considered as truly MRD-negative with great caution and have to be confirmed by FG transcript and/or MFA quantifications.

No relevant conflicts of interest to declare.