Adding an RNA-based gene fusion assay to standard myeloid next-generation sequencing (NGS) can reveal key leukemia-defining mutations that escape traditional cytogenetic methods, according to new research from the University of Michigan. The approach, which identified clinically important but previously undetected gene fusions in acute myeloid leukemia (AML), could improve diagnostic accuracy and influence treatment selection for a subset of patients.
The findings, presented this week at the Association for Molecular Pathology (AMP) annual meeting in Boston, stem from a review of more than 600 AML cases analyzed with an enhanced sequencing panel that included targeted RNA fusion detection. The study was led by Corey Post, MD, of the University of Michigan, who described the work in a poster and presentation at AMP 2025.
AML is a genetically heterogeneous disease, and gene fusions—where segments of different genes become joined—play a central role in defining its molecular subtypes. Some fusions, such as PML::RARA or KMT2A rearrangements, have well-established implications for diagnosis, risk stratification, and targeted therapy. However, conventional karyotyping can miss rearrangements that are cytogenetically “cryptic,” meaning they are not visible under a microscope. These hidden events can delay or obscure accurate classification and, by extension, optimal treatment planning.
To address this diagnostic gap, Dr. Post and colleagues incorporated an RNA-based, probe ligation–based gene fusion assay into their existing Myeloid NGS panel, designed to detect 112 known fusions associated with leukemia. Reviewing all AML and myeloid sarcoma cases sequenced at their institution over a 5-year period, the researchers found gene fusions in 89 of 601 AML samples (14.8%).
Of note, 23 of these fusions—roughly 4% of all AML cases—were cryptic by karyotype and would have gone undetected without the RNA-based approach. “The cryptic rearrangements included 12 cases of AML with NUP98 rearrangement, 6 cases of AML with KMT2A rearrangement, 2 cases of acute promyelocytic leukemia (APL) with PML::RARA fusion, 2 cases of AML with CBFB::MYH11, and 1 case each of AML with DEK::NUP214 rearrangement, AML with KAT6A::CREBBP, and NPM1::MLF1,” Dr. Post reported. Each of these rearrangements defines a distinct disease entity and carries diagnostic or therapeutic significance.
Dr. Post’s team also identified 2 additional AML cases that had suspicious but inconclusive cytogenetic results, later clarified by the RNA assay, and 3 cases in which cytogenetic analysis was unavailable or inadequate. Together, these findings suggest that integrating RNA-based fusion detection into routine myeloid testing could meaningfully expand the diagnostic yield, particularly for cryptic or borderline cases.
The results underscore a growing recognition in hematopathology that RNA-based testing provides a powerful complement to DNA-based sequencing and cytogenetics. This approach aligns with evolving international diagnostic frameworks. Both the 2022 World Health Organization (WHO) classification and the International Consensus Classification (ICC) recognize multiple AML subtypes defined primarily by gene fusions, sometimes regardless of blast count or morphology. Detecting these fusions early, therefore, can have direct implications for how a case is categorized and managed.
The University of Michigan team concluded that at least one method capable of detecting cytogenetically cryptic fusions—whether RNA-based NGS or another validated approach—should be included as part of the comprehensive diagnostic workup for all new AML cases.
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