Mate pair sequencing improves detection of genomic abnormalities in acute myeloid leukemia

Abstract

Objective: Acute myeloid leukemia (AML) can be subtyped based on recurrent cytogenetic and molecular genetic abnormalities with diagnostic and prognostic significance. Although cytogenetic characterization classically involves conventional chromosome and/or fluorescence in situ hybridization (FISH) assays, limitations of these techniques include poor resolution and the inability to precisely identify breakpoints. Method:We evaluated whether an NGS-based methodology that detects structural abnormalities and copy number changes using mate pair sequencing (MPseq) can enhance the diagnostic yield for patients with AML.Results: Using 68 known abnormal and 20 karyotypically normal AML samples, each recurrent primary AML-specific abnormality previously identified in the abnormal samples was confirmed using MPseq. Importantly, in eight cases with abnormalities that could not be resolved by conventional cytogenetic studies, MPseq was utilized to molecularly define eight recurrent AML-fusion events. In addition, MPseq uncovered two cryptic abnormalities that were missed by conventional cytogenetic studies. Thus, MPseq improved the diagnostic yield in the detection of AML-specific structural rearrangements in 10/88 (11%) of cases analyzed. Conclusion:Utilization of MPseq represents a precise, molecular-based technique that can be used as an alternative to conventional cytogenetic studies for newly diagnosed AML patients with the potential to revolutionize the diagnosis of hematologic malignancies. K E Y W O R D Sacute myeloid leukemia, molecular cytogenetics, MPseq

Acute leukemias harboring KMT2A/MLLT10 fusion: a 10‐year experience from a single genomics laboratory

“…Figure illustrates the gene fusion mechanism along with the abnormal FISH signal patterns that were generated. Conventional chromosome, FISH, and MPseq results for patient 18 with discrepant KMT2A BAP (negative) and KMT2A/MLLT10 D‐FISH (positive) results can be visualized in Aypar et al Briefly, MPseq indicated exons 9‐24 of MLLT10 (NM_004641) were inserted into intron 9 of KMT2A (NM_005933) resulting in a KMT2A/MLLT10 fusion.…”

“…To date, the full characterization of KMT2A/MLLT10 gene fusions has heavily relied upon a combination of conventional chromosome, FISH, and reverse transcriptase‐polymerase chain reaction studies . To further characterize 2 cryptic KMT2A/MLLT10 fusions with “normal” chromosomes studies (patients 18 and 48), we performed MPseq, a next‐generation sequencing (NGS)‐based assay that resolves chromosomal rearrangements with significantly higher resolution and precision compared to traditional cytogenetic methodoliges . MPseq results for patient 18 detected an insertion of exons 9‐24 of MLLT10 into intron 9 of KMT2A (illustrated in Aypar et al) and predicts an in frame chimeric KMT2A/MLLT10 fusion.…”

“…To further characterize 2 cryptic KMT2A/MLLT10 fusions with “normal” chromosomes studies (patients 18 and 48), we performed MPseq, a next‐generation sequencing (NGS)‐based assay that resolves chromosomal rearrangements with significantly higher resolution and precision compared to traditional cytogenetic methodoliges . MPseq results for patient 18 detected an insertion of exons 9‐24 of MLLT10 into intron 9 of KMT2A (illustrated in Aypar et al) and predicts an in frame chimeric KMT2A/MLLT10 fusion. This patient case was particularly challenging since the conventional chromosome study and KMT2A BAP results were normal.…”

“…This optimization is made possible during library preparation by the generation of 2‐5 kilobase fragments that have 101 base pairs sequenced on each fragment end, resulting in a lower read depth necessary to detect structural variants in the genome. Detailed protocols and additional information regarding MPseq technology and bioinformatics tools can be found in Drucker et al, Johnson et al, and Aypar et al…”

“…When available, cells were cultured, harvested, and banded utilizing standard cytogenetic techniques according to specimen-specific pro- Detailed protocols and additional information regarding MPseq technology and bioinformatics tools can be found in Drucker et al, 12 Johnson et al, 13 and Aypar et al 14…”