Massively parallel sequencing of tenosynovial giant cell tumors reveals novel CSF1 fusion transcripts and novel somatic CBL mutations

Abstract

Tenosynovial giant cell tumor (TSGCT) is a rare neoplasm. Although surgical resection is the widely accepted primary treatment for TSGCT, recurrences are frequent, and patients’ joint function may be severely compromised. Previous studies reported that CSF1‐COL6A3 fusion genes were identified in approximately 30% of TSGCTs. The aim of our study was to comprehensively clarify the genomic abnormalities in TSGCTs. We performed whole exome sequencing in combination with target sequence validation on 34 TSGCT samples. RNA sequencing was also performed on 18 samples. RNA sequencing revealed fusion transcripts involving CSF1, including novel CSF1‐VCAM1, CSF1‐FN1 and CSF1‐CDH1 fusions, in 13/18 (72%) cases. These fusion genes were validated by chromogenic in situ hybridization. All CSF1 fusions resulted in the deletion of CSF1 exon 9, which was previously shown to be an important negative regulator of CSF1 expression. We also found that 12 (35%) of the 34 TSGCT samples harbored CBL missense mutations. All mutations were detected in exons 8 or 9, which encode the linker and RING finger domain. Among these mutations, C404Y, L380P and R420Q were recurrent. CBL‐mutated cases showed higher JAK2 expression than wild‐type CBL cases (p = 0.013). CSF1 fusion genes and CBL mutations were not mutually exclusive, and both alterations were detected in six of the 18 (33%) tumors. The frequent deletion of CSF1 exon 9 in the fusion transcripts suggested the importance of this event in the etiology of TSGCT. Our results may contribute to the development of new targeted therapies using JAK2 inhibitors for CBL‐mutated TSGCT.

Detection of CSF1 rearrangements deleting the 3′ UTR in tenosynovial giant cell tumors

“…Furthermore, the transcripts of these rearranged CSF1 sequences were more abundant than the two most common usual CSF1 mRNA variants, suggesting a change in post‐transcriptional regulation might be responsible . Most recently, RNA sequencing of a series of 18 tenosynovial giant cell tumors identified similar CSF1 alterations in 16 cases, all involving a deletion of the 3′ UTR and including, among other loci, partners CDH17 , NOTCH2 and 1q21.1, similar to our own findings . Taken as a whole, these studies, together with our own data, support the theory that it is the loss of the negative regulatory 3′ UTR of CSF1 that is the most consistent driving event in tenosynovial giant cell tumors.…”

“…We found CSF1 alterations by FISH in 83% of all examined cases, including 80% of localized and 95% of diffuse‐type tenosynovial tumors in which FISH was successful, but only 60% of tumors of unspecified type, most of which were diagnosed on core biopsy or piecemeal excision. This was slightly higher than the 61%‐76% rearrangement rates reported in earlier series . Among positive cases, the population fraction of cells with CSF1 alterations (rearrangement and/or deletion) ranged from 2.1 to 30.7% (mean 11.9%), which is similar to the 2%‐25% in prior series .…”

“…More recently, CBL mutations were reported in 35% of 34 tenosynovial giant cell tumors evaluated . CBL encodes a RING finger E3 ubiquitin ligase which is involved in downregulation of tyrosine kinase signaling, including CSF1R, and had been shown to be mutated in a variety of hematolymphoid neoplasms …”

“…13 While the most common CSF1 fusion partner has been reported to be COL6A3, identified in 33% of cases, and thought to result in overexpression of full-length CSF1 via promoter swapping, 7 others have reported that the CSF1 breakpoint was downstream of exon 5, and led to the replacement (by alternate sequences) of the 3 0 untranslated region (UTR) in an otherwise intact CSF1. [14][15][16] Thus, it has been proposed that truncation of this region may account for CSF1 overexpression in some cases of tenosynovial giant cell tumor. 15,16 The CSF1 mRNA transcript responsible for production of the secreted CSF1 isoform is notable for containing a long 3 0 UTR, spanning over 2000 nucleotides.…”

“…[14][15][16] Thus, it has been proposed that truncation of this region may account for CSF1 overexpression in some cases of tenosynovial giant cell tumor. 15,16 The CSF1 mRNA transcript responsible for production of the secreted CSF1 isoform is notable for containing a long 3 0 UTR, spanning over 2000 nucleotides. 3 0 UTRs are known targets for posttranscriptional regulation of mRNA expression, often mediated by miRNAs and AU-rich elements (AREs), and CSF1 expression has been shown to be regulated by both of these factors in studies of ovarian cancer.…”