Cryptic KMT2A::AFDN Fusion Due to AFDN Insertion into KMT2A in a Patient with Acute Monoblastic Leukemia

Abstract

Background: KMT2A rearrangements occur in ~10% of acute myeloid leukemia (AML) cases and are critical for classification, risk stratification, and use of targeted therapy. However, insertions involving the KMT2A gene can evade detection using chromosomal analysis and/or fluorescence in situ hybridization (FISH).

Methods: We present a case of a 22-year-old woman with acute monoblastic leukemia harboring a cryptic KMT2A::AFDN fusion identified by RNA sequencing. Initial FISH showed a 3' KMT2A deletion, while conventional karyotyping and the automated bioinformatic pipeline for optical genome mapping (OGM) did not identify the canonical translocation.

Results: To resolve these discrepancies, metaphase KMT2A FISH (break-apart fusion probe) was performed to assess whether KMT2A was translocated to another chromosome. However, the results did not support this possibility. As the fusion signal remained on the normal chromosome 11, with the 5' KMT2A signal localized to the derivative chromosome 11. A subsequent manual review of the OGM data revealed a cryptic ~300 kb insertion of AFDN into the 3' region of KMT2A, reconciling the discrepancies between chromosomal analysis, FISH, and RNA fusion results.

Conclusions: This case highlights the importance of integrating multiple testing modalities with expert review when there is a discrepancy. Our findings emphasize the need for a comprehensive approach to genomic assessment to enhance diagnostic accuracy and guide therapeutic decision-making.

Keywords: KMT2A::AFDN rearrangement; RNA fusion panel; acute monocytic leukemia; fluorescence in situ hybridization (FISH); optical genome mapping.

Figure 1

Morphologic and immunophenotypic findings in peripheral blood and bone marrow specimens. (A,B): Peripheral blood (A) and bone marrow aspirate (B) smears show large blasts with open chromatin, variably conspicuous nucleoli, round to indented nuclear membranes, and moderate basophilic cytoplasm. No Auer rods were identified (×1000). (C): The bone marrow biopsy specimen shows a hypercellular bone marrow with sheets of large blasts displaying a starry-sky appearance (×400). (D): Immunohistochemical analysis shows that the blasts are positive for lysozyme (×400). (E–I): Flow cytometric immunophenotypic analysis shows that the blasts are positive for CD34, CD117, CD4, CD33, CD38, CD64, CD123, HLA-DR, TdT, and MPO (dim, ~5%).

Figure 2

Chromosomal analysis, interphase FISH, RNA fusion panel, and metaphase FISH results. (A): Chromosomal analysis reveals a complex karyotype, 47,XX,+8,del(9)(q21q31)[11]/47,idem,inv(11)(q14q23)[8]. (B): Interphase FISH using a KMT2A break-apart probe shows one intact yellow fusion signal and one green signal, indicating a 3′ KMT2A deletion. (C): RNA fusion panel identifies fusion transcripts between exon 8 of KMT2A and exon 2 of AFDN. (D): Metaphase FISH reveals a normal chromosome 11 with a yellow fusion signal (yellow arrow) and a derivative chromosome 11 with only a 5′ KMT2A green signal (green arrow) and loss of 3′ KMT2A.

Figure 3

OGM results. (A) The OGM circos plot reveals trisomy 8, a deletion on the long arm of chromosome 9, and an interchromosomal translocation between chromosomes 9 and 10. Additionally, within chromosome 11q23, one inversion (marked by a blue dot), one deletion (marked by a red dot), and several intrachromosomal rearrangements are detected. (B) Initial review of OGM near the KMT2A locus highlights a 3′ end deletion of KMT2A, indicated by a thick red arrow and line on the reference chromosome 11, corresponding to the red dot in the circos plot (A). Furthermore, an inversion involving the CBL gene is denoted by a thick blue arrow and line on the reference chromosome 11, corresponding to the blue dot in the circos plot (A). (C) Detailed manual review of the insertion event reveals an insertion encompassing exons 2–11 of the AFDN gene, depicted by yellow bars within the red box on consensus map 2. This pattern of yellow bars matches that observed in (D). (D) Reference map and consensus map of chromosome 4 further illustrate the pattern of the AFDN (exon 2-11)). Throughout these figures, the OGM data are represented with specific visual elements to aid interpretation. The blue lines depict the alignment of the sample’s OGM map data (consensus map) to the reference genome. Within these blue lines, blue bars identify regions of consistent alignment or matched segments, while yellow bars pinpoint regions where structural variations—such as deletions or insertions—have been detected.