Abstract 3018: RET rearrangements as promising therapeutic targets in breast cancer
Authors: Paratala Bhavna S. , Ross Jeffrey S. , Williams Casey B. , Petrosky Whitney , Williams Kirstin A. , Chung Jon , Dolfi Sonia C. , Ganesan Shridar , Ali Siraj , Leyland-Jones Brian , Hirshfield Kim M. 
Published: July 2017

Abstract
Background: Receptor tyrosine kinase alterations have played a significant role in therapeutic decisions for cancer due to their oncogenic nature and response to targeted small molecule kinase inhibitors. Increased genomic profiling of tumors using hybrid-capture based next-generation sequencing approaches now reveal the presence of previously unknown fusions and alterations involving kinases in a diverse set of cancers. Here we report the presence and therapeutic significance of recurrent and novel fusions involving RET, a known oncogenic tyrosine kinase receptor, in breast cancer.
Methods: Comprehensive genomic profiling on formalin-fixed, paraffin embedded patient tumor tissues was performed using FoundationOne platform that covers the entire coding region for 315 cancer-related genes and introns of 28 genes involved in rearrangements at a depth of 500-1000X (Foundation Medicine, MA). Out of 23 rearrangements, two representative RET fusion expression vectors were synthesized and expressed in non-tumorigenic cell lines (breast MCF10A and mouse 3T3 fibroblasts) and were evaluated for RET kinase signaling, drug response, and tumorigenicity.
Results: RET gene fusions, the canonical NCOA4-RET and a novel, noncanonical RASGEF1A-RET fusion, were identified in two separate breast cancers and both include exons required to retain the intact kinase domain of Ret. The novel RASGEF1A-RET fusion includes the non-coding region of RASGEF1A potentially resulting in a truncated RET protein using an alternate internal start site in exon 11 of RET. In vitro characterization of both fusions expressed in mouse 3T3 and human MCF10a cell lines revealed constitutive kinase activation and subsequent downstream signaling as evidenced by phosphorylation of Ret, Erk and Akt. This is the first reported noncanonical RET rearrangement resulting in a 5’ truncated but functional RET kinase. Non-tumorigenic cell lines with stable expression of either rearrangement showed transformed phenotypes assessed by changes in morphology, enhanced growth rate, colony forming ability, and tumor formation in mice. RET fusion-transformed cells were exquisitely sensitive to treatment with RET inhibitors when evaluated in both short-term and long-term functional assays. NCOA4-RET was found by CGP in an index case of metastatic ER+/HER2+ breast cancer that had radiographic evidence of disease progression while on trastuzumab, pertuzumab, and anastrazole. Subsequent treatment with cabozantinib plus anastrazole led to a rapid clinical and radiographic response.
Conclusion: CGP techniques involving hybrid-capture based approaches can identify previously unreported but recurrent RET gene fusions in breast cancer. Here, we show that RET fusions including both canonical and non-canonical complex rearrangements are functional and may represent promising therapeutic targets in selected breast cancer patients.
Citation Format: Bhavna S. Paratala, Jeffrey S. Ross, Casey B. Williams, Whitney Petrosky, Kirstin A. Williams, Jon Chung, Sonia C. Dolfi, Shridar Ganesan, Siraj Ali, Brian Leyland-Jones, Kim M. Hirshfield. RET rearrangements as promising therapeutic targets in breast cancer [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 3018. doi:10.1158/1538-7445.AM2017-3018