Pediatric AML is characterized by numerous genetic aberrations (chromosomal translocations, deletions, insertions) impacting its classification for risk of treatment failure. Aberrations are described by classical cytogenetic procedures (karyotyping, FISH), which harbor limitations (low resolution, need for cell cultivation, cost-intensiveness, experienced staff required). Optical Genome Mapping (OGM) is an emerging chip-based DNA technique combining high resolution (~500 bp) with a relatively short turnaround time. Twenty-four pediatric patients with AML, bi-lineage leukemia, and mixed-phenotype acute leukemia were analyzed by OGM, and the results were compared with cytogenetics. Results were discrepant in 17/24 (70%) cases, including 32 previously unknown alterations called by OGM only. One newly detected deletion and two translocations were validated by primer walking, breakpoint-spanning PCR, and DNA sequencing. As an added benefit, in two cases, OGM identified a new minimal residual disease (MRD) marker. Comparing impact on risk stratification in de novo AML, 19/20 (95%) cases had concordant results while only OGM unraveled another high-risk aberration. Thus, OGM considerably expands the methodological spectrum to optimize the diagnosis of pediatric AML via the identification of new aberrations. Results will contribute to a better understanding of leukemogenesis in pediatric AML. In addition, aberrations identified by OGM may provide markers for MRD monitoring.
Background:Background: AML is the 2nd most frequently diagnosed blood cancer in children affecting approximately 130 patients annually in Germany. Pediatric AML is a heterogeneous malignancy that has multiple genetic aberrations. This is relevant for classification, prognosis and selection of optimal therapy. To detect these alterations established procedures (karyotyping, FISH, RNA-based techniques) are used with well-known limitations (resolution of karyotyping and FISH, respectively; cell cultivation is necessary to generate metaphases; cost-intensiveness; necessity of experienced and intensively trained staff). OGM is an emerging technique addressing these limitations. Based on extraction of ultra-high weight DNA followed by fluorescent labelling at the sequence CTTAAG a barcode-like pattern of the genomic DNA is created. The DNA strands labelled in this way are stretched and drawn as single ultra-long fragments through nanochannels. The results are translated into molecule maps which are compared to the reference genome (resolution down to 500 bp) allowing the identification of genetic alterations. This method combines high resolution with picturing almost whole chromosomes without cell cultivation. Aims: Aims:The aim of this study was to test to what extend OGM might supplement classical cytogenetics (CCG) to detect risk defining genetic aberrations at initial diagnosis in pediatric patients with AML. Methods:Methods: We analyzed 24 specimen of pediatric AML, MPAL and bilineage leukemia patients by OGM starting from stored frozen material (bone marrow, peripheral blood) obtained at initial diagnosis. Results of OGM were compared with karyotyping and FISH. Primer walking and breakpoint spanning PCR were used to validate newly detected aberrations by OGM in selected cases. Applying breakpoint-spanning PCR, validated aberrations were used as markers for assessment of minimal residual disease (MRD) in selected cases during AML treatment. Results:Results: Overall, we detected discrepant results between CCG and OGM in 17/24 (70%) cases including 9 cases in
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