RNA sequencing identifies a novel USP9X-USP6 promoter swap gene fusion in a primary aneurysmal bone cyst.

Primary aneurysmal bone cyst (ABC) is a benign multiloculated cystic lesion of bone that is defined cytogenetically by USP6 gene rearrangements. Rearrangements involving USP6 are promoter swaps, usually generated by fusion of the noncoding upstream exons of different partner genes with exon 1 or 2 of USP6, thus leading to transcriptional upregulation of full-length USP6 coding sequence. Testing for USP6 rearrangements is used diagnostically to distinguish it from secondary ABC and other giant cell-rich primary bone tumors. In this report, we present a case of a 16-year-old male with a primary ABC of the left distal femur. USP6 break apart fluorescence in situ hybridization was positive for a rearrangement and conventional chromosome analysis identified a reciprocal X;17 translocation. In order to identify the putative USP6 fusion partner, we performed RNA sequencing and uncovered a novel USP9X-USP6 promoter swap fusion. This result was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) and by mate pair sequencing thus showing the utility of these alternative methodologies in identifying novel fusion candidates. Ubiquitin-specific protease 9X (USP9X), like USP6, encodes a highly conserved substrate-specific deubiquitylating enzyme. USP9X is highly expressed in a number of tissue types and acts as both an oncogene and tumor suppressor in several human cancers. We conclude that oncogenic activation of USP6 via USP9X promoter exchange represents a novel driver of primary ABC formation.

Clinical Utility Validation of an Automated Ultrarapid Gene Fusion Assay for NSCLC.

Introduction: Gene rearrangements are frequent oncologic drivers in NSCLC, and many are suitable for treatment with Food and Drug Administration-approved or experimental targeted therapies. We evaluated the accuracy, specimen acceptance profile, and limits of detection of a rapid fusion assay (Idylla GeneFusion Assay), a commercially available ultrarapid molecular assay, for its clinical utility. Methods: A collection of 97 specimens which had previously undergone next-generation sequencing testing were analyzed using the rapid fusion assay. Accuracy was evaluated by sensitivity and specificity compared with the next-generation sequencing results. The performance characteristics were tested by using a variety of different clinically relevant specimen types. Limits of detection were assessed by evaluating different input of tumor percentage and material amount. Results: The rapid fusion assay was found to have 100% sensitivity in detecting fusions of ALK, ROS1, RET, NTRK1, and MET exon 14 skipping and 83% sensitivity for NTRK2/3 fusions. There were 100% specificity in detecting fusions of ROS1, RET, NTRK2/3, and MET exon 14 skipping and 98% specificity for ALK. Testing was successful with formalin-fixed paraffin-embedded biopsy and surgical tissues, cell blocks from fine-needle aspiration and pleural fluid (down to 5% tumor content, 18 mm2 tissue scraped), cytology smears (≥300 cells), and previously extracted RNA (minimal 20 ng). Conclusions: The rapid fusion assay is quick, accurate, and versatile, allowing reliable detection of ALK, ROS1, RET fusions, and MET exon 14 skipping in NSCLC, and NTRK fusions. Rapid molecular testing may expedite treatment with appropriate targeted therapies.