MRD Monitoring in AML Patients with MLL-MLLT4 by Quantification of Fusion Gene Transcripts and Genomic Chromosomal Breakpoint Sequences

Abstract

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 FG transcript at RNA/cDNA level in comparison with 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.

Genomic breakpoint-specific monitoring of measurable residual disease in pediatric non-standard-risk acute myeloid leukemia

“…Advantages of DNA-based assays are the more accurate quantification of MRD levels as a surrogate of cell number due to the allele-specific nature of the assay and the highly standardized interpretation of results. 19 , 20 Furthermore, superior stability of DNA allows for reliable MRD quantification even after cross-country shipment of samples and the patient-specific quantification reduces risk of intra-laboratory sample contamination to a minimum.…”

“…Advantages of DNA-based assays are the more accurate quantification of MRD levels as a surrogate of cell number due to the allele-specific nature of the assay and the highly standardized interpretation of results. 19,20 Furthermore, superior stability of DNA allows for reliable MRD quantification even after cross-country shipment of samples and the patient-specific quantification reduces risk of intra-laboratory sample contamination to a minimum. In the present study, our aim was to determine the feasibility of using genomic breakpoint-specific MRD quantification to monitor treatment response in intermediateand high-risk subtypes of pediatric AML prospectively in a routine clinical setting.…”