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Abstract 4700 Statement 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). Materials and methods 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. Results 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. Conclusions 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. Disclosures: No relevant conflicts of interest to declare.

Statement. The MLL-MLLT4 (former MLL-AF6) fusion gene (FG) is relatively rare genetic abnormality, predominantly found in AML. It averages 3–5% among other MLL rearrangements. Here we present data of MRD monitoring in 2 patients with AML carrying an MLL-MLLT4 rearrangement by 2 approaches: using 1. FG transcript at RNA/cDNA level in comparison with 2. FG at genomic DNA level. Materials and methods. Patients (pts) were diagnosed according French-American-Britain (FAB) classification. Initial diagnostics included cytomorphology, immunophenotyping of blast cells, cytogenetics, FISH and reverse transcriptase PCR (RT-PCR). RT-PCR products were directly sequenced afterwards. In both cases identification of genomic chromosomal breakpoint sequences within MLL and MLLT4 genes was done by long-distance inverse PCR (LDI-PCR). MRD quantification in genomic DNA was performed using patient-specific primers and probes by real-time quantitative PCR (RQ-PCR). 500 ng of DNA was used per reaction. Standard curve was received by serial 10-fold dilutions of pts’ DNA into the DNA isolated from pooled lymphocytes of ten healthy donors. b-actin was used as DNA quality and quantity control. Detection of FG transcript kinetics during treatment was performed by RQ-PCR according to “Europe Against Cancer” recommendations for normalization by using control gene ABL (Gabert J. et al Leukemia, 2003, 17) and for using 10-fold dilutions of plasmids carrying MLL-MLLT4 fragment as source of standard curve. MRD value for cDNA targets were estimated as previously described (Beillard E. et al Leukemia, 2003, 17). Each sample was run in triplicates. According to the treatment design, time-points for MRD estimation were scheduled before each block of treatment. Totally 5 samples were evaluated in each patient (initial and 4 follow-ups). FLT3 -ITD status was estimated at the time of diagnosis. Informed consent was obtained in both cases. Patients’ characteristics, treatment and clinical outcome | | Case # 1 | Case #2 | |:-------------------------------------------------------------------:| ----------------------------------------------------------------------------------------- | ------------------------------- | | Age | 58 | 13 | | Sex | Male | Male | | Initial WBC*106/ml | 5.5 | 94.9 | | Immunophenotype | CD34+CD117+HLA-DR+ CD11c+CD13+CD33+CD65+ | CD34+CD117+CD13+ CD33+CD45+MPO+ | | Cytogenetics | 46, XY, del(5)(q?), der(5)t(5;6;11) (q22;q15q27;q23), der(6)t(5;6) (q22;q15), del der(11) | 46, XY, t(6;11)(q27;q23) | | FISH with LSI MLL | MLL deletion | MLL split | | RT-PCR | MLL-MLLT4 positive | MLL-MLLT4 positive | | MLL-MLLT4 FG transcript | exon 9-exon 2 | exon 9-exon 2 | | Localization of genomic chromosomal breakpoint within MLL and MLLT4 | intron 9-intron 1 | intron 9-intron 1 | | FLT3-ITD | Negative | Negative | | Induction treatment | 7+3 | AIE | | Consolidation therapy | 2× HAM 2× HDAC | 1× HAM 1× FLAG 1× HAE | | Maintenance | − | + | | Duration of therapy, months | 8 | 7 | | Achievement of CR | + | + | | OS, months | 8 | 7 | | EFS, months | 6 | 5 | | Current status | Alive in CR | Alive in CR | Results . Despite of achievement of CR, MLL-MLLT4 FG transcripts were detected in every sample tested after induction and consolidation chemotherapy by RQ-PCR. MRD value in case #1 in cDNA was fluctuated significantly within 2 log. Although in case #2 there was successive reduction from 260% at the beginning of treatment till 0.7% before maintenance therapy (after HAE block). Limited dilution series of a MLL-MLLT4-positive RNA into RNA of ten healthy donors showed a sensitivity limitation of 1E–05. For quantification of genomic chromosomal breakpoint sequences b-actin was amplified in each well. Deviation between Ct values of b-actin in different wells did not exceed ±2.0. In case # 1 the standard curve of the RQ-PCR assay for MLL-MLLT4 FG had slope of −3.19. Correlation coefficient was 0.987. Quantitative range of this assay was 1E–04 and sensitivity 1E–5. It was also observed a considerable variation of MRD levels in genomic DNA during treatment, like it was observed in MRD monitoring by FG transcripts. Fluctuations run up to 2.5 log. In case #2 the standard curve of the RQ-PCR assay for MLL-MLLT4 had a slope of −3.63 with correlation coefficient 0.992. Quantitative range of this assay was reached 1E–04 with sensitivity 1E–05. MRD level in this patient constantly decreased. Conclusions . The same tendency has been shown in each patient: fluctuations of MRD levels (2.5 log in case #1) and successive reduction (case #2). Results received at RNA/cDNA level and in genomic DNA cannot substitute each other, but they can be used as additives. It has been demonstrated that quantification of MLL-MLLT4 FG in genomic DNA is precise and suitable for MRD monitoring.