Abstract P6-04-03: In-depth genomic analysis of acquired resistance to multiple sequential lines of endocrine therapy in breast cancer
Authors: Turnbull Arran K , Xa Youli , Martinez-Perez Carlos , Oikonomidou Olga , Meehan James , Gray Mark , Carey Lisa A , Perou Charles M , Dixon J Michael 
Published: March 2020

Background: 80% of all breast cancers (BCs) are ER+. Not all respond to endocrine therapy (ET) and many eventually develop resistance. An in-depth genomic analysis of cancers that acquired resistance (aR) to multiple sequential lines of ET after an initial period of response has been performed.

Patients: A unique cohort of tissues from 20 post-menopausal women with ER+ BC was collected. All patients initially responded to neoadjuvant ET; 19/20 received an aromatase inhibitor (AI): letrozole (LET) (n=18) or anastrozole (AN) (n=1), 1 patient received fulvestrant followed by adjuvant tamoxifen (TAM) (mean treatment duration 22 months, range 4-67) before developing resistance. Patients were then treated by surgery or 2nd line ET. Overall, 13/20 received 2nd line ET either AN (n=1), TAM (n=10) or exemestane (EX) (n=1); 6 went on to receive 3rd line ET with EX (n=5) or LET (n=1).

Methods: Serial RNA & DNA from 3-5 cancer samples per patient (89 samples) had Ribo0-RNAseq and DNA exome sequencing. Somatic mutations and copy number alterations (CNA) were determined. Fisher’s exact test was used to compare mutation frequencies (MFs). Differential gene expression analysis was performed using two-class unpaired Significance Analysis of Microarrays (SAM).

Results:

Mutations: Comparisons of MFs between all sensitive vs resistant tumours identified mutations in SAAL1 and SLC9A9 as being enriched in sensitive tumours while mutations in GATA3 and ZFPM2 were enriched across tumours with acquired resistance to ET. Only KMT2C was identified as enriched in resistant tumours when comparing sensitive to only those that acquired resistance to 1st line LET. When comparing sensitive tumours to only those that had acquired resistance to multiple lines of ET (LET and TAM +/- AN and EX), sensitives were significantly enriched for mutations in CCDC141 and SLC9A9 and multi-drug resistant (MDR) tumours were significantly enriched for mutations in 11 genes including ESR1 and GATA3, and genes involved in cell adhesion (CDH1, FLG, FLG2 and FREM2). ESR1 mutations were identified in 6/20 patients; 5 patients had D538G mutations, 2 appeared during resistance to 1st line LET and 3 in MDR tumours. 1 patient had an ESR1 frame-shift mutation which appeared during resistance to 1st line LET. GATA3 was mutated in 7/20 patients, of which 4 had mutations in all samples including baseline, 2 had mutations uniquely present in MDR tumours and 1 had mutations that appeared during resistance to 1st line LET.

CNA: No significant gains or losses were identified when comparing sensitive tumours to those resistant to 1st line LET alone, but significant gains were identified across multiple chromosomes in MDR tumours. Gain of ESR1 was seen in 8/20 patients; in 3 patients there was ESR1 gain at diagnosis which persisted through initial response and subsequent aR. 5 patients developed ESR1 gain during MDR but not during initial resistant to 1st line LET.

Gene Expression: Analysis comparing sensitive and MDR tumours identified 82 differentially expressed genes (FDR=0). MDR tumours had higher proliferation and higher expression of transcriptional regulators including FOS and FOSB, histone cluster genes and genes involved in oxidative phosphorylation. MDR tumours also had higher expressions of GATA3 induced genes.

Discussion: In contrast to 1st line ET resistance, aR to multiple lines was characterised by significant gains across multiple chromosomes including ESR1 gain in a quarter of tumours. MDR tumours were also characterised by enrichment of mutations in particular genes including ESR1 (D538G in particular) which occurs in one-third of patients with aR. GATA3 is expressed in &gt;90% of BCs, is reported to be mutated in up to 10% of all BCs may be integral to the functions of ESR1; mutations in GATA3 were enriched in MDR tumours and may highlight a new area of ET resistance.

Citation Format: Arran K Turnbull, Youli Xa, Carlos Martinez-Perez, Olga Oikonomidou, James Meehan, Mark Gray, Lisa A Carey, Charles M Perou, J Michael Dixon. In-depth genomic analysis of acquired resistance to multiple sequential lines of endocrine therapy in breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-04-03.