(P 355) FUSION TRANSCRIPTOME PROFILING DEFINES THE MONOCLONAL NATURE OF MULTIFOCAL EPITHELIOID HEMANGIOMA OF BONE

Objective: Epithelioid hemangioma (EH) of bone is a highly controversial entity. Indeed, despite a benign histological appearance, EH of bone may show lymph node involvement and multifocal presentation. Whether multifocal lesions represent independent tumors or metastatic foci is still debated. Although tumor multifocality and lymph node involvement would suggest an aggressive clinical behavior, this is apparently contradicted by some clinical studies. EHs of bone are hallmarked by FOS or FOSB gene rearrangements that may be used in differential diagnosis with EH mimics. In this work we retrospectively analyzed 42 patients treated in a single institution from 1978 to 2021, to describe EH clinical behavior and define the clonal relationships between multifocal lesions, using gene fusion breakpoints as a clonality marker.

Methods: Fusion-targeted analysis was performed using a customized Archer FusionPlex RNA-sequencing panel, supplemented with spike-ins primers for FOS and FOSB. Libraries were run on an Illumina MiSeq sequencing platform. The Archer Analysis bioinformatics suite and in-house-optimized fusion callers (Arriba and Fusion Catcher) were used for fusion transcript identification.
Whole transcriptome analysis was performed by RNA-sequencing using the Illumina Stranded Total Ribo-Zero Plus RNA Library Prep kit. Libraries were run on an Illumina Hiseq 1000 platform. PCA and unsupervised hierarchical clustering were performed on the top 5000 genes with the highest variance.
RT-PCR and Sanger sequencing were used for orthogonal validations.

Results: Prognosis of our series of EH of bone was excellent. Multifocal synchronous presentation was detected in 17 of 42 patients (40%). Patients were treated with curettage (57%), resection (29%) or biopsy followed by radiotherapy or embolization (14%). Only 5 local recurrences were detected and no death of disease.
To clarify whether multifocal EHs represent multicentric disease or metastases, multiple lesions of 3 cases were molecularly profiled. Synchronous EHs from the same patient showed a very similar transcriptional profile and expressed the same fusion transcript (involving FOS or FOSB) with identical gene breakpoints at both RNA and DNA level, demonstrating a common clonal origin.

Conclusion: Our results indicate that EH is a tumor with an excellent prognosis. Molecular analysis of synchronous EH lesions indicates that they are clonally related and therefore represent metastasis of a common original clone. This finding is in apparent contradiction to the benign clinical course of the disease. We propose that tumor multiplicity in EH of bone likely reflects a phenomenon of passive spreading, whereby EH tumor cells, which are facilitated in their capacity to disseminate because of their vascular nature, retain their benign biological nature.