Abstract PS16-05: Identification of pathogenic RET alterations in cell-free DNA (cfDNA) from patients with metastatic breast cancer
Authors: Taylor Mary Love , Mayro Benjamin , Drusbosky Leylah , Anders Carey , Force Jeremy 
Published: February 2021

Background: RET is an important proto-oncogene involved in the development of various cancers for which we have FDA approved therapies. RET alterations are a known mechanism of resistance against breast cancer systemic therapies and higher RET expression has been demonstrated in breast cancer brain metastases compared to their primary tumors. While the genomic characterization of RET alterations has occurred from bulk breast tumors, the frequency and type of RET alterations in metastatic breast cancer (MBC) have not been fully characterized from cfDNA. The purpose of this study was to identify the incidence of RET genomic alterations occurring in cfDNA from patients with MBC and elucidate which RET alterations may increase RET kinase activity or may function as mechanisms of resistance against the FDA approved RET inhibitor, selpercatinib.

Methods: We queried 16,053 reports from Guardant Health between June 2015 - October 2019 to identify the incidence of RET alterations detected in cfDNA from MBC. We classified each alteration type into the following categories: fusions, single nucleotide variants (SNVs), or indels. Focus was placed on characterizing RET SNVs. Amino acid changes occurring at conserved regions across multiple species were identified. We compared known activating mutations in EGFR and ERBB2 with homologous regions in RET. In silico modeling with PyRx was used to dock selpercatinib onto the RET kinase (PDB 6NJA). Three-dimensional in silico analyses with ChimeraX was utilized to further determine which alterations may increase RET kinase activity or may induce resistance against selpercatinib.

Results: Nonsynonymous RET alterations from the Guardant Health breast cancer database were found in 162 samples from a cohort of 16,053 patients indicating an overall incidence of 1.0%. Alterations included: 3 (1.9%) RET-CCDC6 fusions, 2 (1.2%) RET-KIF5B fusions, 6 (3.7%) indels, and 151 (93.2%) SNVs. Of the 151 samples with SNVs, we identified 37 (23%) and 63 (38.9%) point mutations occurring in the transmembrane/juxtamembrane and kinase domains, respectively, and 77 occurred at highly conserved regions across species. The most frequent hotspot mutations occurring in 4 or more unique breast cancers included H594P, 6 (4.0%); S696L, 4 (2.6%); C634Y/G, 5 (3.3%); R813W/Q, 4 (2.6%); and M918T, 8 (5.3%). We aligned RET with homologous kinases and found 8 (5.3%) RET mutations (R721Q, V804L, M868I, R873Q, G894S) and (A641T, S891L, D925H) that corresponded to known activating mutations in EGFR (E709A/K, T790M, R831H, R836C, G857V) and ERBB2 (V659E, T862A/S, R896C), respectively. Three-dimensional analyses indicate that RET alterations occurring within the hinge (E805Q), HRD (H872R, R873Q), and DFG (D892Y, G894S) regions, and regulatory spine (D933N) of the RET kinase are postulated to induce resistance against selpercatinib.

Conclusions: We found that a modest incidence of RET genomic alterations occur in cfDNA from patients with MBC. Novel somatic alterations in RET were identified from Guardant Health that were not detected in the public domain. A portion of RET SNVs occurred at highly conserved regions across species or at known homologous kinase activating mutations suggesting these specific RET mutations may increase RET kinase activity and might be actionable therapeutic targets in breast cancers harboring these mutations. Three dimensional analyses of the RET protein tyrosine kinase further illustrate which specific alterations may increase RET kinase activity or induce resistance against selpercatinib.

Citation Format: Mary Love Taylor, Benjamin Mayro, Leylah Drusbosky, Carey Anders, Jeremy Force. Identification of pathogenic RET alterations in cell-free DNA (cfDNA) from patients with metastatic breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS16-05.