Background: The menin inhibitor revumenib was recently FDA approved for treating patients with relapsed or refractory KMT2A-rearranged (rKMT2A) acute leukemias. Cytogenetics, FISH, and targeted next-generation sequencing (NGS) frequently miss KMT2A 11q23 partial tandem duplications (KMT2A-PTD) ). Although KMT2A-PTDs have expression signatures similar torKMT2A, they were excluded from revumenib’s registration trial. Preclinical models have shown that menin inhibitors may also be effective for KMT2A-PTD, highlighting the need for precise breakpoint and KMT2A fusion product detection. Here, we evaluated the effectiveness of high-resolution WGS to identify a diverse array of KMT2A-PTD.

Methods: Using a WGS assay (Tempus xH) optimized for comprehensive profiling of myeloid neoplasms, we capture the entire KMT2A locus at base pair resolution. DNA was extracted from blood or bone marrow aspirates and was used to construct paired-end libraries via tagmentation. Sequencing was performed on the Illumina NovaSeq-X platform, achieving a mean coverage of 80X. Data were analyzed using the DRAGEN Platform with custom post-processing filters. Exon copy number calls from a targeted NGS assay and exon capture RNAseq NGS assay (Tempus xT and xR, respectively) were used for verification.

Results: WGS from 230 hematopoietic neoplasms (68% AML, 18% MDS, 12% CML, and 2%others) identified 13 specimens (5.6%) containing a KTM2A-PTD, with variant allele frequencies(VAFs) between 9-66%. All PTDs contained breakpoints within known intron boundaries: one breakpoint in intron 1 (13/13) with terminal breakpoints located in intron 8 (6/13) or intron 10(7/13). RNA data was available for 11 of 13 specimens and contained direct support for the presence of all the KMT2A-PTDs (100%). Using an NGS-targeted panel, exon-level copy calls were assessed for all 13 specimens with PTDs. Although unvalidated, we observed exon level gains in 9 of the 13 specimens (69%). As shown in prior studies, KMT2A-PTDs were mutually exclusive to other translocations, including rKMT2A. However, other high-frequency mutations for myeloid disease were present in select samples including mutations in IDH1, DNTM3A,WT1, and RUNX1.

Conclusions: WGS is an effective tool for detecting KMT2A alterations that may be missed by traditional techniques such as NGS targeted capture, FISH or cytogenetics. 100% concordance was observed between WGS and RNA seq for KMT2A-PTDs, supporting the reliability of WGS.The FDA approval of menin inhibitors for KMT2A-rearranged AML/ALL suggests potential clinical opportunities for broad tests (WGS) to identify other rearrangements, including KMT2A-PTDs, highlighting the need for further research into targeted anti-leukemia therapies.

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