63. Comparative analysis of testing methods used for the detection of internal tandem duplications in the KMT2A/MLL gene

Abstract

The Lysine (K)-Specific Methyltransferase 2A (KMT2A) gene (formerly MLL) that resides on human chromosome 11q23 is frequently rearranged in de novo and therapy related leukemia. KMT2A translocations with various partner genes are often easily detected by classic molecular and cytogenetic approaches. However, the detection of small intragenic insertions, the partial tandem duplications of the KMT2A gene (KMT2A-PTD), is challenging. Additional co-occurring alterations, such as Trisomy 11 which alerts one to the PTD, may further hamper the identification of KMT2A-PTD. KMT2A-PTDs occur in 3-10% of adult AML and the presence of a KMT2A-PTD is associated with a poorer AML outcome. As such, accurate detection of KMT2A-PTDs in AML routine clinical diagnostic workup is important. Here we assessed the ability of three orthogonal technologies for KMT2A-PTD analysis:1) an hybridization-capture based Next Generation Sequencing targeting the KMT2A gene region, 2) an MLPA based assay covering exons 4 and 36 of KMT2A and 3) Optical Genome Mapping. We analyzed tumor samples of ten AML patients with KMT2A-PTD and evaluated the relative performance, strengths and weaknesses of these technologies. KMT2A-PTDs are in-frame duplications that typically encompass exons 3 through 9. However, our results revealed the presence of unsuspected variants of KMT2A. Specifically, copy number based approaches, in the absence of structural data, can lead to false positive and negative calls due to genomic alterations, such as unbalanced translocations, involving KMT2A. Therefore, our data suggest that a combined structural and copy number approach are required to accurately detect KMT2A PTDs.