Abstract P4-09-07: UltraSEEK breast cancer panel for low frequency mutation detection in breast cancer brain metastasis
Authors: Giannoudis Athina , Eastoe Lee , Charlton Christopher , Hickson Nicholas , Zakaria Rasheed , Platt-Higgins Angela , Rudland Philip S , Irwin Darryl , Sartori Alexander , Jenkinson Michael D , Palmieri Carlo 
Published: March 2020

Abstract
Background: Breast cancer brain metastases (BCBM) are a growing clinical problem and a significant cause of morbidity and mortality. The genomic landscape of BCBM can be divergent from that of the primary breast cancer (BC) and extracranial metastasis. Identifying key and targetable genomic aberrations within BCBM will be key in developing novel therapeutic strategies to treat this area of unmet and to need improve patient outcomes. Identifying rare and low-level mutations due to tumour heterogeneity in cancer samples and/or poor quality DNA isolated from formalin-fixed paraffin-embedded (FFPE) tissues requires tools that offer both low frequency detection and efficient use of starting material. This study aims to assess paired primary BC and brain metastasis (BM) for aberrations within known BC drivers: TP53, PIK3CA, ERBB2, ESR1 and AKT utilising the MassARRAY® UltraSEEK™ technology (Agena Bioscience, San Diego, USA), that provides a targeted, multiplexed method for detecting rare events and with sensitivity as low as 0.1%, developed to support DNA isolated from FFPE tissues, plasma and cerebrospinal fluid.
Methods: DNA was isolated (Qiagen GeneRead FFPE kit) from of 32 BC and their matched BM cases and screened using the custom UltraSEEK™ BC Panel (Agena Bioscience, San Diego, USA). The Panel tests 44 mutations across 5 oncogenes in 8 multiplex assays; AKT1 (E17K, L52R), ERBB2 (G309E, G309A, S310F, L755R, L755S, L755_T759del, D769H, D769Y, V777L, V777L, L869R), ESR1 (A283V, K303R, E380Q, V392I, S436P, V534E, L536R, L536Q, L536Q, Y537N, Y537S, Y537C, D538G, S576L), PIK3CA (N345K, C420R, E542K, E545K, E545Q, E545A, H1047R, H1047L, E542K), TP53 (R175H, R213*, Y220C, R248W, R248W, R248Q, R273C, R273H).
Results: Preliminary data analysis using the automated Typer software v4.0.26.74 (Agena Bioscience) identified more than one mutations in 29/32 (90.6%) primary BCs and 26/32 (81.3%) BM. TP53, ESR1, PIK3CA were mutated in 82.7%, 41.4% and 27.6% respectively of primary cases and in 80.8%, 34.6% and 23.1% respectively of BCBM. Of note, the pattern of mutations was similar in only 13/32 (40.6%) of the paired cases. The commonest mutations identified in both the primary and BCBM were TP53 (R175H, R273H, R248Q, R213X), PIK3CA (H1047L, E545K, E542K). The ESR1 (S576L) was the commonest ESR1 mutation identified in both BC and BM but in only one paired case, whereas the Y537N was present in only 2 BM cases (2/9, 22.2% of BM cases harbouring ESR1 mutations) cases. In addition, the AKT1-E17K was present in 3 patients; a paired case of BC and BM, one BC and one BM. The ERBB2-L755S was present in 1 BC and the ERBB2-L755_T759del only in a BM. As this is a custom BC panel, further analysis and validation is required.
Conclusion: This study provides further evidence that the genomic landscape of the BCBM can differ from the primary, that BCBM contain potential actionable mutations and that they harbour ESR1 mutations. Such BCBM specific genomic data will be key to developing rationale therapeutic studies to develop treatments for this area of unmet need. The UltraSEEK™ technology provides a powerful tool to investigate low abundance mutations without compromising analytical accuracy and sensitivity.
Citation Format: Athina Giannoudis, Lee Eastoe, Christopher Charlton, Nicholas Hickson, Rasheed Zakaria, Angela Platt-Higgins, Philip S Rudland, Darryl Irwin, Alexander Sartori, Michael D Jenkinson, Carlo Palmieri. UltraSEEK breast cancer panel for low frequency mutation detection in breast cancer brain metastasis [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 P4-09-07.