CIC rearrangements have been reported in two-thirds of EWSR1-negative small blue round cell tumors (SBRCTs). However, a number of SBRCTs remain unclassified despite exhaustive analysis. Fourteen SBRCTs lacking driver genetic events by RNA sequencing (RNAseq) analysis were collected. Unsupervised hierarchical clustering was performed using samples from our RNAseq database, including 13 SBRCTs with non-CIC genetic abnormalities and 2 CIC-rearranged angiosarcomas among others. Remarkably, all 14 study cases showed high mRNA levels of ETV1/4/5, and by unsupervised clustering most grouped into a distinct cluster, separate from other tumors. Based on these results indicating a close relationship with CIC-rearranged tumors, we manually inspected CIC reads in RNAseq data. FISH for CIC and DUX4 abnormalities and immunohistochemical stains for ETV4 were also performed. In the control group, only 2 CIC-rearranged angiosarcomas had high ETV1/4/5 expression. Upon manual inspection of CIC traces, 7 of 14 cases showed CIC-DUX4 fusion reads, 2 cases had DUX4-CIC reads, while the remaining 5 were negative. FISH showed CIC break-apart in 7 cases, including 5 cases lacking CIC-DUX4 or DUX4-CIC fusion reads on RNAseq manual inspection. However, no CIC abnormalities were detected by FISH in 6 cases with CIC-DUX4 or DUX4-CIC reads. ETV4 immunoreactivity was positive in 7 of 11 cases. Our results highlight the underperformance of FISH and RNAseq methods in diagnosing SBRCTs with CIC gene abnormalities. The downstream ETV1/4/5 transcriptional up-regulation appears highly sensitive and specific and can be used as a reliable molecular signature and diagnostic method for CIC fusion positive SBRCTs.

In recent years, undifferentiated small round cell sarcomas (USRCSs) have been divided into a variety of new, rare, sarcoma subtypes, including the group of Ewing-like sarcomas, which have the morphological appearance of Ewing sarcomas, but carry CIC-DUX4, BCOR-CCNB3 and other gene fusions different from the classic EWSR1-ETS gene fusion. Using high-throughput RNA-sequencing (RNA-seq) analyses, we identified a novel recurrent gene fusion, CRTC1-SS18, in two cases of USRCS that lacked any known translocation. RNA-seq results were confirmed by reverse transcription polymerase chain reaction, long-range polymerase chain reaction, and fluorescence in situ hybridization. In vitro, we showed that the cells expressing the gene fusion were morphologically distinct and had enhanced oncogenic potential as compared with control cells. Expression profile comparisons with tumours of other sarcoma subtypes demonstrated that both cases clustered close to EWSR1-CREB1-positive tumours. Moreover, these analyses indicated enhanced NTRK1 expression in CRTC1-SS18-positive tumours. We conclude that the novel gene fusion identified in this study adds a new subtype to the USRCSs with unique gene signatures, and may be of therapeutic relevance. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

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Undifferentiated pleomorphic sarcoma of bone (UPSb) is a rare primary bone sarcoma that lacks a specific line of differentiation. There is very little information about the genetic alterations leading to tumourigenesis or malignant transformation. Distinguishing between UPSb and other malignant bone sarcomas, including dedifferentiated chondrosarcoma and osteosarcoma, can be challenging due to overlapping features. To explore the genomic and transcriptomic landscape of UPSb tumours, whole‐exome sequencing (WES) and RNA sequencing (RNA‐Seq) were performed on UPSb tumours. All tumours lacked hotspot mutations in IDH1/2 132 or 172 codons, thereby excluding the diagnosis of dedifferentiated chondrosarcoma. Recurrent somatic mutations in TP53 were identified in four of 14 samples (29%). Moreover, recurrent mutations in histone chromatin remodelling genes, including H3F3A, ATRX and DOT1L, were identified in five of 14 samples (36%), highlighting the potential role of deregulated chromatin remodelling pathways in UPSb tumourigenesis. The majority of recurrent mutations in chromatin remodelling genes identified here are reported in COSMIC, including the H3F3A G34 and K36 hotspot residues. Copy number alteration analysis identified gains and losses in genes that have been previously altered in UPSb or UPS of soft tissue. Eight somatic gene fusions were identified by RNA‐Seq, two of which, CLTC‐VMP1 and FARP1‐STK24, were reported previously in multiple cancers. Five gene fusions were genomically characterised. Hierarchical clustering analysis, using RNA‐Seq data, distinctly clustered UPSb tumours from osteosarcoma and other sarcomas, thus molecularly distinguishing UPSb from other sarcomas. RNA‐Seq expression profiling analysis and quantitative reverse transcription‐polymerase chain reaction showed an elevated expression in FGF23, which can be a potential molecular biomarker for UPSb. To our knowledge, this study represents the first comprehensive WES and RNA‐Seq analysis of UPSb tumours revealing novel protein‐coding recurrent gene mutations, gene fusions and identifying a potential UPSb molecular biomarker, thereby broadening the understanding of the pathogenic mechanisms and highlighting the possibility of developing novel targeted therapeutics. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

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