Anchored multiplex PCR for targeted next-generation sequencing reveals recurrent and novel USP6 fusions and upregulation of USP6 expression in aneurysmal bone cyst.

Primary aneurysmal bone cyst (ABC) is a neoplastic process due to recurrent translocations involving the USP6 gene. By fluorescence in situ hybridization, up to 69% of primary ABCs harbored USP6 translocations; no USP6 translocation was found in secondary ABC or giant cell tumor of bone (GCT). GCT can recur locally, metastasize to the lungs in some cases, and rarely undergo malignant transformation. Differentiating primary ABC from its mimics is important for treatment and prognosis. We evaluated USP6 fusion and expression in 13 cases of primary and 1 case of secondary ABC, and 9 cases of GCT using nucleic acid extracted from formalin-fixed, paraffin-embedded tissue and a next generation sequencing (NGS)-based assay. USP6 fusions including 7 novel fusions and USP6 transcripts were identified in all 13 primary ABCs. Nine cases with strong evidence of fusions showed high levels of USP6 transcripts by reverse transcription-PCR (RT-PCR). The remaining four had no detectable USP6 expression by a first-round of RT-PCR but the presence of USP6 transcripts was identified by a second-round, nested PCR. The major fusions were confirmed by RT-PCR followed by Sanger sequencing. No USP6 fusion or transcript was detected in any of the GCTs or the case of secondary ABC by NGS or by two rounds of PCR. All USP6 translocations resulted in fusion of the entire USP6 coding sequence with promoters of the fusion gene leading to upregulation of USP6 transcription, which is likely the underlying mechanism for ABC oncogenesis. © 2016 Wiley Periodicals, Inc.

The NAB2-STAT6 gene fusion in solitary fibrous tumor can be reliably detected by anchored multiplexed PCR for targeted next-generation sequencing.

Solitary fibrous tumor (SFT) is a mesenchymal tumor of fibroblastic origin, which can affect any region of the body. 10-15% of SFTs metastasize and metastatic tumors are uniformly lethal with no effective therapies. The behavior of SFT is difficult to predict based on morphology. Recently, an intrachromosomal gene fusion between NAB2 and STAT6 was identified as the defining driving genetic event of SFT and different fusion types correlated with tumor histology and behavior. Due to the proximity of NAB2 and STAT6 on chromosome 12, this fusion may be missed by fluorescence in-situ hybridization. We evaluated 12 SFTs from 10 patients. All tumors showed strong nuclear staining for STAT6 by immunohistochemistry (IHC). The same formalin-fixed, paraffin-embedded blocks for IHC were used for gene fusion detection by a next-generation sequencing (NGS)-based assay. Targeted RNA fusion sequencing for gene fusions was performed using the Universal RNA Fusion Detection Kit, the Archer(™) FusionPlex(™) Sarcoma Panel and the Ion Torrent PGM, and data were analyzed using the Archer Analysis Pipeline 3.3. All tumors were positive for NAB2-STAT6 fusion. Six types of fusions were detected: NAB2ex4-STAT6ex2, NAB2ex2-STAT6ex5, NAB2ex6-STAT6ex16, NAB2ex6-STAT6ex17, NAB2ex3-STAT6ex18 and NAB2intron6-STAT6Ex17. The NGS findings were confirmed by RT-PCR followed by Sanger sequencing. No STAT6 fusion was detected in selected morphologic mimics of SFT. The assay also allows for detection of novel fusions and can detect NAB2-STAT6 fusions at a single-base resolution.