Abstract 490: Identification of oncogenic gene fusions in primary colon cancers
Authors: Braak Robert R. Coebergh van den , Kloosterman Wigard P. , Pieterse Mark , Roosmalen Markus van , Sieuwerts Anieta M. , Stangl Christina , Brunekreef Ronne , Lalmahomed Zarina S. , Ooft Salo , Galen Anne van , Smid Marcel , Lefebvre Armel , Zwartkruis Fried J. , Martens John W. , Foekens John A. , Biermann Katharina , Koudijs Marco J. , IJzermans Jan N. , Voest Emile E. 
Published: July 2017

Abstract
Introduction: Fusion genes (such as ALK-EML) are potentially attractive targets for anti-cancer treatment. In the era of personalized medicine exploring the identification, incidence, and functionality of fusion genes may contribute to effective treatment approaches.
Methods: We performed a comprehensive and unbiased screening for gene fusions in a clinically well-defined prospectively collected cohort of 278 primary stage I to III colon cancers. Illumina RNA sequencing was performed using RNA from the fresh frozen samples. The STAR fusion gene detection pipeline and GATK RNA-seq variant calling were used to identify fusion genes and detect somatic genetic variations. Gene fusions were considered relevant when recurrent, when resulting in divergent expression (outlier analysis) or when functional relevance was predicted (i.e. kinase fusions).
Results: 2.5% of all samples contained a relevant gene fusion. Kinase fusions were most prevalent with a frequency of 1.8%. Three BRAF-fusions were identified, two known (TRIM24-BRAF & AGAP3-BRAF) and one novel fusion (DLG1-BRAF). Co-expression with ERK1 in HEK293 cells resulted in enhanced EKR1 phosphorylation. The outlier analysis revealed four unique fusions (EML4-NTRK3, RRBP1-RET, USPX9-ERAS & EIF3E-RSPO2). The EML4-NTRK3 fusion led to increased expression of the tyrosine kinase encoding domain exons, which is retained in the fusion transcript. Co-expression with ERK1 in HEK293 cells resulted in enhanced EKR1 phosphorylation. The tumor with the RRBP1-RET fusion was pan negative for known driver mutations, as reported in literature. Mate-pair sequencing of the USPX9-ERAS fusion gene region revealed that the fusion gene was caused at the genomic level by a highly local chromothripsis event on chromosome X spanning solely the region covered by USP9X and ERAS, leading to high ERAS expression. ERAS is a constitutionally active RAS protein and normally only expressed in embryonic stem cells. Analysis of phosphorylated AKT after expression of the USP9X-ERAS fusion gene in NIH-3T3 A14 cells showed activation of AKT signaling. The occurrence of one R-spondin fusion in our set (0.4%) is low compared to the previously described 9.5%, which may be explained by difference in incidence of KRAS- or BRAF-mutations in the two cohorts, since all R-spondin fusions occurred in a tumor with a KRAS- or BRAF-mutation. All fusion genes were expressed in cell lines resulting in the activation of AKT signaling. Finally, oncogenic fusions were only found in lymph node negative tumors and were not observed in tumors with classical driver mutations in BRAF and KRAS except for the R-spondin fusion.
Conclusions: We found oncogenic gene fusions including novel fusions with a frequency of 2.5%, including an USP9X-ERAS fusion with strong oncogenic activity in vitro. Although recurrent fusion genes are rare events in colorectal cancer they may represent functional drivers and provide potential novel leads for personalized therapeutic strategies.
Citation Format: Robert R. Coebergh van den Braak, Wigard P. Kloosterman, Mark Pieterse, Markus van Roosmalen, Anieta M. Sieuwerts, Christina Stangl, Ronne Brunekreef, Zarina S. Lalmahomed, Salo Ooft, Anne van Galen, Marcel Smid, Armel Lefebvre, Fried J. Zwartkruis, John W. Martens, John A. Foekens, Katharina Biermann, Marco J. Koudijs, Jan N. IJzermans, Emile E. Voest. Identification of oncogenic gene fusions in primary colon cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 490. doi:10.1158/1538-7445.AM2017-490