Genomic and transcriptomic characterisation of undifferentiated pleomorphic sarcoma of bone

Abstract

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.

Keywords: CNV; FGF23; RNA sequencing; Whole exome sequencing; chromatin remodelling genes; gene expression and hierarchical clustering analyses; gene fusions; sarcomas; undifferentiated pleomorphic sarcoma of bone.

Figure 1.. The mutational and gene fusion landscape of UPSb.

(A) Recurrent genes identified by whole exome sequencing in 14 tumours, grouped according to their biological pathway and function. (B) Gene fusions identified by RNA sequencing in eight tumours. CCCG: COSMIC Cancer Census Gene; DGIdb: The Drug Gene Interaction database. Gene described ‘cancer driving gene’ in CCGC or druggable in DGIdb are shaded in grey. †: TRIO is classified ‘cancer driver gene’ by IntOGen.

Figure 2.. Recurrent somatic mutations in TP53, H3F3A, ATRX and DOT1L.

All the mutations (black balls) are missenses except for one an in-frame insertion in H3F3A. The relative positions of mutations are shown in the predicted protein sequence of (A) TP53, (B) H3F3A, (C) ATRX and (D) DOT1L. T.A.: p53 transactivation domain; T.M.: p53 tetramerization domain; ADD: ATRX-DNMT3-DNMT3L domain; EZH2: enhancer zeste homologue 2 protein (EZH2) interacting region; DAXX: death domain-associated protein interacting domain; ATPase: Helicase adenosine triphosphatase domain; Catalytic DOT1 domain: Histone-lysine N-methyltransferase DOT1 domain; STAT1: signal transducer and activator of transcription 1 binding motifs.

Figure 3.. The impact of the CLTC-VMP1 and FARP1-STK24 gene fusions on protein domain organization.

The grey shaded area represents the retained protein domains of the fused exons of (A) CLTC-VMP1 and (B) FARP1-STK24. The gene fusion breakpoints are denoted by a black double slash. The CLTC-VMP1 is out of frame, reaching a premature stop (denoted by *) at codon 399 of VMP1 gene. The FARP1-STK24 is in-frame, resulting from joining the first 88 and 211 amino acids of FARP1 and STK24, respectively. ATP B.S: ATP binding site.

Figure 4.. Unsupervised and supervised analyses of UPSb, other sarcomas and SRP090849 datasets using RNA-Seq data.

(A) Unsupervised clustering analysis using Ward’s distance and 1-pearson correlation highlighted two groups of UPSb tumors (dark grey). UPSb_G1 samples are clearly separated from classical UPS (light grey) and synovial sarcomas from the SRP057793 dataset and from osteosarcomas from the SRP090849 dataset. UPSb_G2 samples are closer to classical UPS but remain clearly distinct. (B) Supervised analysis showing a violin plot of the FGF23 gene expression across UPSb and the different tumor types present in the SRP057793 dataset and in the osteosarcoma SRP090849 dataset. Individual samples are shown in circle and group median is represented as a black bar. (C) David Gene Ontology analyses of specific UPSb genes indicating a strong enrichment of immune response genes. Fold enrichment (bar chart) and the –log₁₀ of the hypergeometric test P-value corrected by Bonferroni (black line with closed circles) are shown.