Abstract P3-07-02: Are we missing actionable targets in breast cancer? Novel insights into recurrent Ret alterations
Authors: Hirshfield KM , Paratala BS , Hindoyan A , Dolfi SC , Yilmazel B , Schrock A , Gay L , Ali SM , Ross JS , Williams CB , Nair P , Ganesan S , Leyland-Jones B 
Published: March 2017

Background: Recurrent gene fusions in breast cancer have been rarely reported suggesting that they either are not present or are not easily detected by standard sequencing methods. Comprehensive genomic profiling (CGP) by hybrid capture-based, high depth next-generation sequencing approaches, can be used to detect recurrent rearrangements and other genomic alterations involving target genes. We found that CGP can identify recurrent alterations involving RET, a known oncogenic tyrosine receptor kinase, in a subset of breast cancer.

Methods: CGP using FoundationOne platform was performed interrogating the entire coding region for up to 315 cancer-related genes and introns of up to 28 genes involved in rearrangements at a depth of 500-1000X in formalin-fixed, paraffin embedded tumor tissue (Foundation Medicine, MA). Engineered representative RET fusion vectors were synthesized and expressed in non-tumorigenic cell lines (breast MCF10A and mouse 3T3 fibroblasts), and cells were evaluated for RET kinase signaling, drug response, and tumorigenicity. Patient-derived xenografts (PDX) generated from two triple negative breast cancers (TNBCs) were used in an ex vivo assay (Response3DXTM, Molecular Response LLC, San Diego, CA).

Results: Twenty-two RET rearrangements were identified in 8119 (0.27%) breast cancer cases. Of these, 5 rearrangements were activating RET fusions including CCDC6-RET (n=4) and NCOA4-RET (n=1), that have been described in other cancer types. Five other cases had clear evidence of genomic rearrangement involving RET, but the 5' partners could not be definitively identified. The remaining twelve cases had complex rearrangements of RET including internal duplications. RET amplification was also observed, both in TNBC and in a HER2+ breast cancer at onset of resistance to HER2-targeted therapy.

Both NCOA4-RET and a novel RASGEF1A-RET fusion were characterized in vitro. Non-tumorigenic cells engineered to stably overexpress either RET fusions demonstrated transformed phenotypes. The fusions were constitutively active, as shown by endogenous phosphorylation of the kinase domain, and drove activation of downstream signaling as shown by increased phosphorylation of ERK and AKT. Cells transformed by RET-fusions were exquisitely sensitive to treatment with RET inhibitors. Interestingly, a PDX model of RET-amplified TNBC was sensitive to treatment with a PIK3CA inhibitor. An index case of ER+/PR-/HER2+, metastatic breast cancer that had radiographic evidence of disease progression while on trastuzumab, pertuzumab, and anastrazole was found to have a NCOA4-RET fusion by CGP. Subsequent treatment with with cabozantinib plus anastrazole led a rapid clinical and radiographic response.

Conclusions: CGP can identify recurrent RET rearrangements in breast cancer that act as primary oncogenic drivers and can be therapeutically targeted. RET alterations may also play a role in acquired resistance to HER2-targeted therapies, suggesting a role for combined RET and HER2-targeted therapy in this setting. Our data demonstrate that RET alterations can be identified by clinical-grade CGP and are promising candidates as therapeutic targets in selected breast cancer patients.

Citation Format: Hirshfield KM, Paratala BS, Hindoyan A, Dolfi SC, Yilmazel B, Schrock A, Gay L, Ali SM, Ross JS, Williams CB, Nair P, Ganesan S, Leyland-Jones B. Are we missing actionable targets in breast cancer? Novel insights into recurrent Ret alterations [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-07-02.