A RUNX1: RUNX1T1 AML with a simultaneous false positive KMT2A rearrangement: FISH interpretation pitfalls.

Introduction: KMT2A rearrangement (KMT2Ar) is a common genomic alteration in acute leukemia that can be effectively targeted by menin inhibitors. While FISH is the standard laboratory test for KMT2Ar, false positives can occur.

Case report: We present a case of AML in which both RUNX1::RUNX1T1 and KMT2Ar were identified by karyotype analysis and FISH. Although a targeted RNA next generation sequencing (NGS) assay confirmed the presence of the RUNX1::RUNX1T1 fusion, it did not detect a KMT2A fusion transcript. To investigate the discrepancy between the positive KMT2A FISH result and the negative fusion transcript, we performed whole-genome mate-pair DNA NGS to examine the KMT2A locus on chromosome 11q23. This analysis revealed a breakpoint located 5.8 kb downstream of KMT2A, which did not disrupt the gene itself. Given that KMT2A FISH probes cover approximately 1 Mb around KMT2A, this subtle shift led to a split-apart signal pattern mimicking a genuine KMT2A rearrangement, resulting in a false positive FISH interpretation.

Conclusion: This case highlights a false positive KMT2Ar in primary AML, indicating the need for additional molecular testing for confirmation.