Transcriptome sequencing to detect gene fusions in cancer

Abstract

Recurrent gene fusions, typically associated with haematological malignancies and rare bone and soft-tissue tumours, have recently been described in common solid tumours. Here we use an integrative analysis of high-throughput long- and short-read transcriptome sequencing of cancer cells to discover novel gene fusions. As a proof of concept, we successfully used integrative transcriptome sequencing to 're-discover' the BCR-ABL1 (ref. 10) gene fusion in a chronic myelogenous leukaemia cell line and the TMPRSS2-ERG gene fusion in a prostate cancer cell line and tissues. Additionally, we nominated, and experimentally validated, novel gene fusions resulting in chimaeric transcripts in cancer cell lines and tumours. Taken together, this study establishes a robust pipeline for the discovery of novel gene chimaeras using high-throughput sequencing, opening up an important class of cancer-related mutations for comprehensive characterization.

Fig 1. Employing massively parallel sequencing to discover chimeric transcripts in cancer

a, Schema representing our approach to employ transcriptome sequencing to identify chimeric transcripts. ‘Long read’ sequences compared with the reference database are classified as ‘Mapping’, ‘Partially Aligned’, and ‘Non-Mapping’ reads. Partially aligning reads are considered putative chimeras and are categorized as inter- or intra-chromosomal chimeras. Integration with short read sequence data is utilized for short-listing candidate chimeras and assessing the depth of coverage spanning the fusion junction. b, “Re-discovery” of TMPRSS2-ERG fusion on chromosome 21. Short reads (Illumina) are overlaid on the corresponding long read (454) represented by colored bars. Sequences spanning the fusion junction are indicated by the partition in the short reads. Chromosomal context of the fusion genes is represented by colored bars punctuated with black lines. Inset displays histogram of qRT-PCR validation of the TMPRSS2-ERG transcript.

Fig 2. Representative gene fusions characterized in the prostate cancer cell line VCaP

a, Schematic of USP10-ZDHHC7 fusion on chromosome 16. Exon 1 of USP10 (red) is fused with exon 3 of ZDHHC7 (green), located on the same chromosome in opposite orientation. Inset displays histogram of qRT-PCR validation of USP10-ZDHHC7 transcript. b, Schematic of a complex intra-chromosomal rearrangement leading to two gene fusions involving HJURP on chromosome 2. Exon 8 of HJURP (red) is fused with exon 2 of EIF4E2 (green) to form HJURP-EIF4E2. Exon 25 of INPP4A (blue) is fused with exon 9 of HJURP (red) to form INPP4A-HJURP. Insets display histograms of qRT-PCR validation of HJURP-EIF4E2 and INPP4A-HJURP transcripts.

Fig 3. Schematic of MIPOL1-DGKB gene fusion in the prostate cancer cell line LNCaP

MIPOL1-DGKB is an inter-chromosomal gene fusion accompanying the cryptic insertion of ETV1 locus (red) on chromosome 7 into the MIPOL1(purple) intron on chromosome 14. Previously determined genomic breakpoints (black stars) are shown in DGKB and MIPOL1. An insertion event results in the inversion of the 3’ end of DGKB and ETV1 into the MIPOL1 intron between exons 10 and 11. Inset displays histogram of qRT-PCR validation of the MIPOL1-DGKB transcript.

Fig. 4. Discovery of the recurrent SLC45A3-ELK4 chimera in prostate cancer and a general classification system for chimeric transcripts in cancer

a, Upper panel, schematic of the SLC45A3-ELK4 chimera located on chromosome 1. Middle panel, qRT-PCR validation of SLC45A3-ELK4 transcript in a panel of cell lines. Inset, histogram of qRT-PCR assessment of the SLC45A3-ELK4 transcript in LNCaP cells treated with R1881. Lower panel, histogram of qRT-PCR validation in a panel of prostate tissues-benign adjacent prostate, localized prostate cancer (PCA) and metastatic prostate cancer (Mets). ETS family gene rearrangement status (by FISH) indicated by horizontal colored bars below graph. Grey not determined (ND); yellow, ETS negative; orange, ETS positive. Horizontal bracket indicates three different metastatic tissues from the same patient (Met4). Asterisk (*) denotes an ETV1 positive sample. b, Chimera classification schema (described in the text).