TERT gene rearrangement in chordomas and comparison to other TERT-rearranged solid tumors

Highlights

• Chordomas are rare, slow-growing neoplasms thought to arise from the foetal notochord remnant. A limited number of studies that examined the mutational profiles in chordomas identified potential driver mutations, including duplication in the TBXT gene (encoding brachyury), mutations in the PI3K/Akt signaling pathway, and loss of the CDKN2A gene. Most chordomas remain without clear driver mutations, and no fusion genes have been identified thus far. We discovered a novel TERT in-frame fusion involving RPH3AL (exon 5) and TERT (exon 2) in the index chordoma case. We screened a discovery cohort of 18 additional chordoma cases for TERT gene rearrangement by FISH, in which TERT rearrangement was identified in one additional case. In our independent, validation cohort of 36 chordomas, no TERT rearrangement was observed by FISH (total 2/55, 3.6%). Immunohistochemistry optimized for nuclear TERT expression showed at least focal TERT expression in 40/55 (72.7%) chordomas. Selected cases underwent molecular genetic profiling, which showed low tumor mutational burdens (TMBs) without obvious driver oncogenic mutations. We next examined a cohort of 1,913 solid tumor patients for TERT rearrangements, and TERT fusions involving exon 2 were observed in 7/1,913 (0.4%) cases. The seven tumors comprised five glial tumors, and two poorly differentiated carcinomas. In contrast to chordomas, the other TERT-rearranged tumors were notable for higher TMBs, frequent TP53 mutations (6/7) and presence of other driver oncogenic mutations, including a concurrent fusion (TRIM24-MET). In conclusion, TERT gene rearrangements are seen in a small subset (2/55, 3.6%) of chordomas. In contrast other TERT-rearranged tumors, where the TERT rearrangements are likely passenger events, the possibility that TERT protein overexpression representing a key event in tumorigenesis is left open.

Abstract

Chordomas are rare, slow-growing neoplasms thought to arise from the foetal notochord remnant. A limited number of studies that examined the mutational profiles in chordomas identified potential driver mutations, including duplication in the TBXT gene (encoding brachyury), mutations in the PI3K/AKT signaling pathway, and loss of the CDKN2A gene. Most chordomas remain without clear driver mutations, and no fusion genes have been identified thus far. We discovered a novel TERT in-frame fusion involving RPH3AL (exon 5) and TERT (exon 2) in the index chordoma case. We screened a discovery cohort of 18 additional chordoma cases for TERT gene rearrangement by FISH, in which TERT rearrangement was identified in one additional case. In our independent, validation cohort of 36 chordomas, no TERT rearrangement was observed by FISH. Immunohistochemistry optimized for nuclear TERT expression showed at least focal TERT expression in 40/55 (72.7%) chordomas. Selected cases underwent molecular genetic profiling, which showed low tumor mutational burdens (TMBs) without obvious driver oncogenic mutations. We next examined a cohort of 1,913 solid tumor patients for TERT rearrangements, and TERT fusions involving exon 2 were observed in 7/1,913 (0.4%) cases. The seven tumors comprised five glial tumors, and two poorly differentiated carcinomas. In contrast to chordomas, the other TERT-rearranged tumors were notable for higher TMBs, frequent TP53 mutations (6/7) and presence of other driver oncogenic mutations, including a concurrent fusion (TRIM24-MET). In conclusion, TERT gene rearrangements are seen in a small subset (2/55, 3.6%) of chordomas. In contrast to other TERT-rearranged tumors, where the TERT rearrangements are likely passenger events, the possibility that TERT protein overexpression representing a key event in chordoma tumorigenesis is left open.

Introduction

Chordomas are rare, slow-growing neoplasms thought to arise from the fetal notochord [1]. Histologically, cords and lobules of cells separated by fibrous septa, often with extensive myxoid stroma, and the “physaliferous” tumor cells are notable for abundant, bubbly and eosinophillic cytoplasm. This unique morphology may be related to their genetics; recurrent mutations have been reported in the LYST gene, which encodes for a lysosomal trafficking regulator [2]. Mutations in the LYST gene have been associated with Chediak-Higashi syndrome, where the mutations result in defective lysosomal biogenesis and enlarged organelles in cytotoxic T-cells [3]. A similar phenomenon may be happening in chordoma cells, which are also notable for abundant vacuoles, which have been characterized as lysosome-related organelles [4]. Possible role for these vacuoles in chemo-resistance has been hypothesized, while they have also been hypothesized as potential therapy targets [4]. Immunohistochemically, the tumors are generally immunoreactive for the S100 protein expression, with brachyury being commonly employed as a diagnostic marker. While brachyury expression is seen in nearly all cases of chordomas and their pro-oncogenic tissue notochord, duplication of the TBXT gene (T-box transcription factor T, encoding brachyury) has been reported to be present in only 3/11 (27%) chordoma cases, suggesting different mechanisms are likely responsible for the brachyury expression in other chordomas. In the rare, familial chordomas focal germline tandem TBXT gene duplication has been reported [5]. Despite these interesting genotype-phenotype correlations, the mechanism of tumorigenesis in chordoma is poorly understood. Loss of CDKN2A (encoding p16(INK4A) and p14(ARF) proteins) and mutations in the PI3K/AKT signalling pathway have been reported in a subset of chordomas, but a large proportion of chordomas remain without clear driver mutations [2].

Telomere length stabilization is an important hallmark of cancer cells, crucial for supporting continued cell division [6]. Telomeres are not necessarily long, and telomere lengths are heterogeneous across the different cancers [7]. Telomerase activation and alternative lengthening of telomeres (ALT) are the two key mechanisms in telomere maintenance. The TERT gene encodes a component of the telomerase complex, namely the telomerase reverse transcriptase [8]. The TERT gene is transcriptionally silent in most non-neoplastic tissues, while aberrant telomerase reactivation is commonly observed, perhaps in up to 90% of human cancers [9]. Telomerase reactivation may be associated with point mutations in the TERT gene (including TERT promoter mutations such as C228T and C250T), TERT gene rearrangements (including TERT fusion transcripts) and TERT DNA copy number gains [7, [10], [11], [12], [13]]. TERT promoter mutation analysis and immunohistochemistry for ATRX (loss of which is associated with telomerase dysfunction) are important components of diffuse glioma workup [14, 15]. However, the TERT gene poses a challenge for molecular pathology laboratories due to the GC-rich nature of the region, and the TERT promoter is not included in many commercial and non-commercial cancer sequencing panels. The TERT gene is also not routinely included in a number of fusion transcript assays.

In this study we identified a case of chordoma with a novel TERT gene fusion gene by RNA sequencing. We screened two cohorts of chordomas by fluorescent in-situ hybridization (FISH), and 2/55 cases were found to harbor TERT rearrangement. Furthermore, we assessed 1,913 non-chordoma, solid tumors by RNA sequencing to assess the frequency of TERT fusions, and we compared the molecular features of TERT-rearranged tumors.