Identification of Novel Fusion Genes in Testicular Germ Cell Tumors

Abstract

Testicular germ cell tumors (TGCT) are the most frequently diagnosed solid tumors in young men ages 15 to 44 years. Embryonal carcinomas (EC) comprise a subset of TGCTs that exhibit pluripotent characteristics similar to embryonic stem (ES) cells, but the genetic drivers underlying malignant transformation of ECs are unknown. To elucidate the abnormal genetic events potentially contributing to TGCT malignancy, such as the existence of fusion genes or aberrant fusion transcript expression, we performed RNA sequencing of EC cell lines and their nonmalignant ES cell line counterparts. We identified eight novel fusion transcripts and one gene with alternative promoter usage, ETV6. Four out of nine transcripts were found recurrently expressed in an extended panel of primary TGCTs and additional EC cell lines, but not in normal parenchyma of the testis, implying tumor-specific expression. Two of the recurrent transcripts involved an intrachromosomal fusion between RCC1 and HENMT1 located 80 Mbp apart and an interchromosomal fusion between RCC1 and ABHD12B. RCC1-ABHD12B and the ETV6 transcript variant were found to be preferentially expressed in the more undifferentiated TGCT subtypes. In vitro differentiation of the NTERA2 EC cell line resulted in significantly reduced expression of both fusion transcripts involving RCC1 and the ETV6 transcript variant, indicating that they are markers of pluripotency in a malignant setting. In conclusion, we identified eight novel fusion transcripts that, to our knowledge, are the first fusion genes described in TGCT and may therefore potentially serve as genomic biomarkers of malignant progression.

ScaR - A tool for sensitive detection of known fusion transcripts: Establishing prevalence of fusions in testicular germ cell tumors

“…These findings support our previous results that showed that RCC1-ABHD12B expression, but not CLEC6A-CLEC4D expression, was significantly reduced when a pluripotent embryonal carcinoma cell line (NTERA2) was differentiated in vitro (14). These observations support a biological significance of these fusion transcripts being markers of pluripotent TGCTs.…”

“…These results indicates that the failure to detect the investigated fusion transcripts in normal GTEx samples is not due to differences in sequencing power between the cohorts, and that these fusion transcripts are specifically present in TGCT and not in normal tissue of the testis. This is in accordance with previously published experimental RT-PCR data (14), although then from relatively few samples.…”

“…There were a median of 42.8 million pairs of reads (min: 27.8 million -max: 132.2 million) with read length of 76 x 2 bp (Table S2). Paired-end RNA-seq data from the ES cell line Shef3, as described in Hoff et al (14), was used together with simulated RNA-seq data from synthetic fusion transcripts for benchmarking (see Benchmarking and data simulation).…”

“…To compare the performance of our scaffold alignment approach to that of established de novo fusion finders, we applied it together with deFuse v.0.7.0 (20) and FusionCatcher v.0.99.5a (21) on the external TGCT and normal testis data sets from TCGA and GTEx. We searched for the fusion transcripts RCC1-ABHD12B, RCC1-HENMT1, CLEC6A-CLEC4D and EPT1-GUCY1A3, as previously identified and characterized in TGCT by Hoff et al (14). The Unix command line tool grep was also applied as a simple blunt tool for comparison to our scaffold approach.…”

Determining the impact of uncharacterized inversions in the human genome by droplet digital PCR

“…Recently, it was shown that this technique could also be used to phase variants separated by up to 200 Kb (Regan et al 2015), which has already been applied to fusion transcript detection (Hoff et al 2016) or to phase deletions into haplotypes (Boettger et al 2016). Here, we have developed new ddPCR assays to genotype quickly and reliably human polymorphic inversions flanked by large IRs, and thanks to the genotype data we demonstrate that most of these inversions are recurrent and that inversion alleles are associated to gene expression changes.…”