Molecular and Cellular Pathobiology Identification of Novel Fusion Genes in Testicular Germ Cell Tumors

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. Cancer Res; 76(1); 108–16. 2015 AACR. Introduction Testicular germ cell tumors (TGCT) are the most common cancer in young men, ages 15 to 44 years (1). Although it is a highly treatable cancer type, exemplified by a 10-year net survival rate of 98% in England and Wales (2), the disease affects men in their prime and treatment can lead to substantially increased morbidity, including cardiovascular disease, reduced fertility, and secondary cancers (3).Histologically, there are twomain subtypes of TGCTs, seminomas, and nonseminomas. Both are thought to develop from the preinvasive stage termed intratubular germ cell neoplasia (IGCN; also known as carcinoma in situ). Nonseminomas are further divided into the pluripotent embryonal carcinomas (EC) and more differentiated subtypes, with either somatic (teratoma) or extraembryonic differentiation (yolk sac tumors, YST, and choriocarcinomas; ref. 4). EC cells are highly similar to embryonic stem (ES) cells, derived from the inner cell mass of the blastocyst stage embryo (5). Both cell types exhibit pluripotent characteristics phenotypically and in gene expression profiles (6, 7). Upon extended passaging in vitro, ES cells have been shown to acquire genetic changes similar to those seen in malignant transformation in vivo of TGCTs and EC, including gain of geneticmaterial from chromosomes 12, 17, and X (8). Gain of chromosome arm12p, often as an isochromosome, i(12p), is found in virtually all cases of TGCT (9, 10). Crucially, despite these similarities, EC cells are malignant in character, whereas ES cells are not. Comparative studies between the two cell types may therefore be useful for characterization of cancerspecific differences in a pluripotent context (5, 11). One such study revealed that several transcription factors located on 12p are overexpressed in EC cells as compared to ES cells (6). Although 12p material is gained in virtually all cases of TGCT, no clear genetic driver for TGCT malignant transformation has been pinpointed (12, 13). Recently, whole-exome sequencing studies have revealed that the number of nonsynonymous mutations in coding regions of the TGCT genome are few, on a scale similar to that of pediatric cancers (14–16). A number of pediatric cancers with a low mutational load are frequently found to harbor fusion genes with oncogenic properties. Examples are MLL rearrangements in acute lymphoblastic leukemia (17), and subtypes of sarcomas classified by distinct chromosomal translocations (18). In Ewing sarcoma, fusions involving EWSR1 are pathognomonic, while the mutation rate is low, estimated at 0.15/Mb of coding sequence (19). In this study, we have performed RNA sequencing of EC cell lines and their nonmalignant counterpart, ES cell lines. Application of a fusion gene analysis pipeline led to the identification of nine novel fusion genes and transcripts, to our knowledge the first described in TGCT. Department of Molecular Oncology, Institute for Cancer Research, Oslo University Hospital-Norwegian Radium Hospital, Oslo, Norway. Centre for Cancer Biomedicine, University of Oslo, Oslo, Norway. Department of Biomedical Science, University of Sheffield,Western Bank, Sheffield, United Kingdom. Centre for Stem Cell Biology, University of Sheffield,Western Bank, Sheffield, United Kingdom. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Corresponding Author: Rolf I. Skotheim, Oslo University Hospital, Norwegian Radium Hospital, P. O. Box 4953, Nydalen, Oslo 0424, Norway. Phone: 47-22781727; Fax: 47-2278-1745; E-mail: [email protected] doi: 10.1158/0008-5472.CAN-15-179