Abstract P4-07-07: Establishing whole-exome sequencing for breast cancer patient care
Authors: Roberts Kate , Leo Paul J , Khoo Jeremy , Febery Alice , Ellis Jonathan , Clout Mhairi , Wheeler Lawrie , Anderson Lisa , Brown Matthew , Bennett Ian 
Published: March 2020

Abstract
Background. Next-generation sequencing (NGS) technology enables profiling of individual tumours and to measure the increasing number of biomarkers relevant to the management of breast cancer patients. The Australian Translational Genomics Centre (ATGC) is a collaboration between the healthcare sector (Metro South Hospital and Health Service), higher education sector (Queensland University of Technology) and a government-run pathology service (Pathology Queensland) to provide genomic profiling of breast cancer patients at the Princess Alexandra Hospital. The program was developed to integrate the ordering, processing and interpretation of large-scale NGS into clinical practice.
Methods. Patients were consented for somatic testing and ordering was integrated into the hospital’s electronic ordering system. Samples were derived from fresh tissue biopsies after surgeries (47%) or from FFPE histology sections. No selection criteria were applied during the initial phase and the cohort was representative of newly presenting patients at the hospitals breast cancer clinic. Patients were sequenced using a NATA-accredited ISO15189 program using whole-exome sequencing (WES) combined with a high-coverage spike-in panel of known cancer genes. Clinical reports included the calculation of tumour purity, tumour mutational burden (TMB), the assessment of copy number events and somatic mutations down to 3% allele frequency.
Standard molecular testing in Australia includes ER, PR and HER2 status, and additional testing included testing of Tier 1-2 somatic variants in the genes ABCC3, AKT1, CCND1, CCNE1, CDKN2A, ERBB2, ESR1, FGF3, FGFR1, FGFR2, MTOR, NCOA3, NF2, PIK3CA, PIK3R1, PTEN, RB1, RSF1, SF3B1, TP53. Results. Seventy-one patients were tested by WES/panel, and an average of 1.5 clinically significant Tier 1- 2 mutations were detected per patient. In 77% of cases, the molecular profiling could stratify patients to those with either PI3K/Akt/mTOR pathway activation (by PIK3CA activating mutations, AKT, MTOR mutations or PTEN loss) or CDK4/6 activation (by CCND1 expansion or CDKN2A loss). The most frequently observed mutations were PIK3CA activation (40%) and CCND1 copy number expansions (24%). A small proportion (n=3) were found to have mTOR or TCS1/2 mutations reported to have association with a durable response to mTOR inhibitors. Additionally, 11 patients (15%) had a high TMB (TMB, &gt;6.8 mutations/megabase) with 6 having &gt;10 mutations/Mb. Within this high-TMB cohort, 3 were found to be ER-PR-HER-, however, the majority (n=7) were ER+PR+HER2- patients. Calculation of the tumour purity indicated that, despite expert resection of the biopsies to isolate the most tumour dense regions, tumour purity was not significantly enriched. In 58% of cases, the tumour purity was less than 50%, and in 13% of samples, it was less than 25%, indicating that in clinical practice the method of sequencing must be robust, as many samples have significant amounts of contaminating stroma.
Conclusion. We demonstrate that integration of WES/panel testing into clinical practice is practical and provides multiplexed testing of current and emerging biomarkers in a significant number of tested patients. While a high proportion of patients had mutations that could stratify them to targeted therapies in the event of metastatic disease, the somatic molecular profiles did not modify first line therapy decision-making. Training of clinical staff for patient consent and the dissemination of findings, the development of a dedicated molecular tumour board, and decision protocols to identify patients of metastatic risk were identified as key developments in this clinical program. The program identified previously unidentified subsets of patients including a subset of ER+ patients with high TMB for which for there are no effective treatments option in Australia.
Citation Format: Kate Roberts, Paul J Leo, Jeremy Khoo, Alice Febery, Jonathan Ellis, Mhairi Clout, Lawrie Wheeler, Lisa Anderson, Matthew Brown, Ian Bennett. Establishing whole-exome sequencing for breast cancer patient care [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 P4-07-07.