Abstract P5-04-14: Tracking ESR1 mutation clonal evolution in breast cancer using in situ mutation detection
Authors: Martinez-Perez C , Turnbull AK , Tanioka M , Fernando A , Renshaw L , Keys J , Wheless A , Garrett A , Parker J , He X , Sims AH , Carey LA , Perou CM , Dixon JM 
Published: May 2019

Abstract
Background: Approximately 70% of breast cancers (BCs) are estrogen receptor positive (ER+). Not all ER+ cancers respond to endocrine therapy (ET) and many eventually develop acquired resistance. Next-generation sequencing (NGS) has shown ESR1 mutations (ESRMs) are present in 10-50% of recurrent/metastatic cancers treated with aromatase inhibitors (AIs). Many of these mutations are located in the ligand-binding domain of ER, so they can lead to constitutive activation. This suggests ESRMs are a major mechanism of acquired resistance to endocrine therapy (ET) and numerous studies have shown a link between ESRMs and reduced sensitivity to 2nd line ET. The aim of this project was to investigate the incidence and clonal evolution of common ESRMs in BCs resistant to multiple sequential ETs using NGS, as well as novel PCR and in situ mutation detection methods.
Methods: We have optimised an allele-specific real-time PCR (rtPCR) assay and an in situ mutation detection method (ER-ISMD) for the assessment of ESRMs. Both have been designed to identify a missense gain-of-function D538G mutation with a single nucleotide-resolution in formalin-fixed paraffin-embedded (FFPE) BC tissues.
Two unique cohorts have been studied:
(A) 20 post-menopausal women (PMW) with ER+ BC who acquired resistance to letrozole and were treated with up to 4 subsequent lines of ET. Serial RNA and DNA from 3-5 cancer samples per patient (58 samples from 20 patients) were analysed by Ribo0-RNAseq, DNA exome sequencing, rtPCR and ER-ISMD.
(B) 150 PMW with ER+ BC who developed local (n=79), lymph node (n=59) or distant (n=12) recurrences on 1st line adjuvant letrozole, anastrozole or tamoxifen. Of these, 48 patients developed subsequent recurrences on 2nd line ET. Tissue samples from each recurrence and matched primary BC were collected.
Results: In cohort A, 5/20 patients (20%) had expansion of a D538G ESR1 mutation clone at time of resistance 1st line ET (3:letrozole, 1:anastrozole, 1:tamoxifen). The mutant allele frequency (MAF) increased further in the 4 BCs treated with 2nd line ET (2:tamoxifen, 2:exemestane) and further still in the 1 BC who received 3rd line exemestane. 0/6 patients with ESRM responded to subsequent ET. Allele-specific rtPCR and ER-ISMD have been used to validate these findings and also identified low frequency ESRM clones in the sequential samples prior to the development of clinical resistance, that were not reported by NGS. Both methods have also been applied to screen tissues from patients in cohort B, where ESRMs have also been identified in recurrent samples. Complete analysis is currently ongoing.
Conclusions:
· ESRMs develop and expand in some BCs as a mechanism for acquired resistance to ET and are associated with a lack of response to subsequent standard ETs.
· Allele-specific rtPCR can detect ESRMs and is more cost-effective and easier to use than NGS for ER mutation analysis.
· Some ESRMs predate clinical resistance.
· ER-ISMD is a novel approach that allows for identification and visualisation of the distribution of mutant clones in morphologically intact FFPE tissue.
· ER-ISMD has the potential to become a clinically useful tool to help direct the use of 2nd line ET in routine care.
Citation Format: Martinez-Perez C, Turnbull AK, Tanioka M, Fernando A, Renshaw L, Keys J, Wheless A, Garrett A, Parker J, He X, Sims AH, Carey LA, Perou CM, Dixon JM. Tracking ESR1 mutation clonal evolution in breast cancer using in situ mutation detection [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-04-14.