Assessment of HMGA2 and PLAG1 rearrangements in breast adenomyoepitheliomas

Abstract

Breast adenomyoepitheliomas (AMEs) are rare epithelial-myoepithelial neoplasms that may occasionally produce myxochondroid matrix, akin to pleomorphic adenomas (PAs). Regardless of their anatomic location, PAs often harbor rearrangements involving HMGA2 or PLAG1. We have recently shown that the repertoire of somatic genetic alterations of AMEs varies according to their estrogen receptor (ER) status; whilst the majority of ER-positive AMEs display mutually exclusive PIK3CA or AKT1 hotspot mutations, up to 60% of ER-negative AMEs harbor concurrent HRAS Q61 hotspot mutations and mutations affecting either PIK3CA or PIK3R1. Here, we hypothesized that a subset of AMEs lacking these somatic genetic alterations could be underpinned by oncogenic fusion genes, in particular those involving HMGA2 or PLAG1. Therefore, we subjected 13 AMEs to RNA-sequencing for fusion discovery (n = 5) and/or fluorescence in situ hybridization (FISH) analysis for HMGA2 and PLAG1 rearrangements (n = 13). RNA-sequencing revealed an HMGA2-WIF1 fusion gene in an ER-positive AME lacking HRAS, PIK3CA and AKT1 somatic mutations. This fusion gene, which has been previously described in salivary gland PAs, results in a chimeric transcript composed of exons 1-5 of HMGA2 and exons 3-10 of WIF1. No additional in-frame fusion genes or HMGA2 or PLAG1 rearrangements were identified in the remaining AMEs analyzed. Our results demonstrate that a subset of AMEs lacking mutations affecting HRAS and PI3K pathway-related genes may harbor HMGA2-WIF1 fusion genes, suggesting that a subset of breast AMEs may be genetically related to PAs or that a subset of AMEs may originate in the context of a PA.

Fig. 1

Fusion genes involving HMGA2 or PLAG1 and somatic mutations targeting HRAS, PIK3CA, AKT1 and PIK3R1 in breast adenomyoepitheliomas. a Heatmap depicting fusion gene and somatic mutations targeting HRAS Q61, PIK3CA and AKT E17 hotspot loci and PIK3R1 mutations identified in breast adenomyoepitheliomas (AMEs; n = 13). Cases are shown in columns and genes in rows. Hotspot mutations are annotated as per Chang et al. b Representative Sanger sequencing electropherograms of HRAS Q61 and PIK3CA hotspot loci in AM16. c Representative hematoxylin and eosin micrographs of an AME harboring an HMGA2-WIF1 fusion gene (AM16), and micrographs depicting p63 and estrogen receptor expression. Scale bars, 500 μm (upper left), 100 μm (upper right) and 50 μm (middle and lower panels). ER estrogen receptor, FISH fluorescence in situ hybridization, SNV single nucleotide variant, WT wild-type

Fig. 2

HMGA2-WIF1 fusion gene identified in the epithelial and myoepithelial cells of a breast adenomyoepithelioma. a Schematic representation of the HMGA2-WIF1 fusion transcript identified in AM16, including the exons and domains involved. HMGA2 is on the (+) DNA strand and WIF1 on the (−) DNA strand. The breakpoints of the 5’ and 3’ partner genes are represented as black vertical lines. Eight spanning reads were found to cross the genomic breakpoint of the HMGA2-WIF1 chimeric transcript and are depicted aligned to the predicted junction sequence. b Representative hematoxylin and eosin and FISH micrographs of the epithelial and myoepithelial components of AM16 using HMGA2 dual-color break apart probes (red, 5′ HMGA2; green, 3′ HMGA2). aa aminoacid, AcD acidic domain, DBD DNA binding domain, E epithelium, M myoepithelium, SpD spacer domain. Scale bar, 50 μm