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

Area of Science:

• Hematology
• Genomics
• Molecular Diagnostics

Background:

• KMT2A rearrangement (KMT2Ar) is a frequent genomic alteration in acute leukemia.
• Menin inhibitors are effective targeted therapies for KMT2Ar-positive leukemia.
• Fluorescence in situ hybridization (FISH) is the standard diagnostic test for KMT2Ar, but can yield false positives.

Purpose of the Study:

• To investigate a discrepancy between FISH and RNA sequencing results for KMT2Ar in an acute myeloid leukemia (AML) case.
• To identify the underlying cause of a false positive KMT2Ar FISH result.

Main Methods:

• Karyotype analysis and FISH for KMT2Ar detection.
• Targeted RNA next-generation sequencing (NGS) for fusion transcript identification.
• Whole-genome mate-pair DNA NGS to analyze the KMT2A locus at 11q23.

Main Results:

• Karyotype and FISH indicated both RUNX1::RUNX1T1 and KMT2Ar.
• RNA NGS confirmed RUNX1::RUNX1T1 but not a KMT2A fusion transcript.
• Whole-genome DNA NGS revealed a breakpoint 5.8 kb downstream of KMT2A, not disrupting the gene, causing a false positive FISH signal.

Conclusions:

• This case demonstrates a false positive KMT2Ar result in primary AML.
• Confirmatory molecular testing beyond FISH is crucial for accurate KMT2Ar diagnosis.