Genomic Characterization of Partial Tandem Duplication Involving the Gene in Adult Acute Myeloid Leukemia.

Background: Gene rearrangements affecting are frequent in acute myeloid leukemia (AML) and are often associated with a poor prognosis. gene fusions are often detected by chromosome banding analysis and confirmed by fluorescence in situ hybridization. However, small intragenic insertions, termed partial tandem duplication (KMT2A-PTD), are particularly challenging to detect using standard molecular and cytogenetic approaches.

Methods: We have validated the use of a custom hybrid-capture-based next-generation sequencing (NGS) panel for comprehensive profiling of AML patients seen at our institution. This NGS panel targets the entire consensus coding DNA sequence of . To deduce the presence of a KMT2A-PTD, we used the relative ratio of exons coverage. We sought to corroborate the KMT2A-PTD NGS results using (1) multiplex-ligation probe amplification (MLPA) and (2) optical genome mapping (OGM).

Results: We analyzed 932 AML cases and identified 41 individuals harboring a KMT2A-PTD. MLPA, NGS, and OGM confirmed the presence of a KMT2A-PTD in 22 of the cases analyzed where orthogonal testing was possible. The two false-positive KMT2A-PTD calls by NGS could be explained by the presence of cryptic structural variants impacting and interfering with KMT2A-PTD analysis. OGM revealed the nature of these previously undetected gene rearrangements in , while MLPA yielded inconclusive results. MLPA analysis for KMT2A-PTD is limited to exon 4, whereas NGS and OGM resolved KMT2A-PTD sizes and copy number levels.

Conclusions: KMT2A-PTDs are complex gene rearrangements that cannot be fully ascertained using a single genomic platform. MLPA, NGS panels, and OGM are complementary technologies applied in standard-of-care testing for AML patients. MLPA and NGS panels are designed for targeted copy number analysis; however, our results showed that integration of concurrent genomic alterations is needed for accurate KMT2A-PTD identification. Unbalanced chromosomal rearrangements overlapping with can interfere with the diagnostic sensitivity and specificity of copy-number-based KMT2A-PTD detection methodologies.