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

Abstract

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.

Novel ASAP1‐USP6, FAT1‐USP6, SAR1A‐USP6, and TNC‐USP6 fusions in primary aneurysmal bone cyst

“…In our series, all of the 11 consecutive primary ABCs harbored USP6 gene rearrangement, as revealed by NGS. Similarly, Guseva et al reported a series of 13 primary ABCs, all of which harbored USP6 gene rearrangements . Although the number of cases in both series combined is not very large, it is reasonable to conclude that USP6 gene rearrangement is consistently present in all ABCs and that ABCs are defined by the presence of a USP6 fusion.…”

“…Although the number of cases in both series combined is not very large, it is reasonable to conclude that USP6 gene rearrangement is consistently present in all ABCs and that ABCs are defined by the presence of a USP6 fusion. This may be important when a primary ABC needs to be differentiated from its potential mimics, including giant cell tumor, chondroblastoma, osteoblastoma, nonossifying fibroma and rarely malignant tumors, such as telangiectatic osteosarcoma . FISH and RT‐PCR methods may fail to recognize a USP6 gene fusion if the fusion partners are located too close, an inversion of the gene is involved, or the USP6 ‐fusion partner is unknown .…”

“…The neoplastic cells in ABC, which are morphologically spindle, ovoid or wavy, represent only a proportion of all cells in the ABC (from 7% to 82%; median, 27%; in one large study) . A subset of ABCs (17% in two series combined) is predominantly or entirely solid, referred to as solid variant of ABC …”

“…While USP6 gene rearrangement was detected by fluorescent in situ hybridization (FISH) in 59% to 63% of primary ABC, there is strong evidence that it is much more frequent and probably even universally present, as shown by next‐generation sequencing (NGS) based studies . There is a growing list of known fusion partners, of which CDH11 seems to be the most frequent; it was detected in 19% and 38% of ABCs in two series . Other fusion partners identified so far include CNBP , COL1A1 , OMD and THRAP3 , CTNNB1 , E1F1 , FOSL2 , PAFAH1B1 , RUNX2 , SEC31A , STAT3 , SPARC and, most recently, USP9X .…”

“…There is a growing list of known fusion partners, of which CDH11 seems to be the most frequent; it was detected in 19% and 38% of ABCs in two series . Other fusion partners identified so far include CNBP , COL1A1 , OMD and THRAP3 , CTNNB1 , E1F1 , FOSL2 , PAFAH1B1 , RUNX2 , SEC31A , STAT3 , SPARC and, most recently, USP9X . Despite different fusion partners, all USP6 rearrangements seem to function by a promoter swap mechanism, resulting in oncogenic activation of USP6 and subsequent ABC formation .…”