19The application of liquid biopsies in metastatic salivary gland cancer to identify candidate therapeutic targets

Background: Adenoid cystic carcinoma (ACC) is a rare salivary gland cancer with no proven drug therapies and only 40% long term survival with surgery and radiotherapy. The development of new drug treatments and the application of a personalised medicine approach in this poorly understood disease requires molecular profiling of tumour material. Liquid biopsies have the potential to accelerate the discovery process as they are minimally invasive and can easily be performed at multiple time points compared with tumour biopsies. This study evaluated the application of circulating tumour cells (CTCs) and circulating free DNA (cfDNA) as liquid biopsies to identify candidate therapeutic targets in ACC. Methods: From May to Dec 2016, 10 patients with metastatic ACC were prospectively recruited to an ethically approved study. Blood samples were collected whilst off therapy and CTCs were isolated and enumerated by CellSearch (EpCAM immunomagnetic cell surface marker enrichment) and Parsortix (size-based enrichment). CellSearch CTCs were extracted and stored in glycerol at -80 Celsius for subsequent single cell manipulation (DEPArray, Silicon Biosystems). Genomic DNA was isolated from peripheral blood mononuclear cells, tumour DNA from the archived formalin fixed parrafin embedded tumour resection specimens and cfDNA was isolated using the automated QIASymphony platform (Qiagen). Whole genome libraries were prepared (Accel 2S DNA kit, Swift Biosciences) and whole genome paired-end next generation sequencing (NGS) was performed using the illumina MiSeq desktop sequencer for shallow depth (0.1-0.2x) copy number analysis (CNA). Bioinformatic analysis was performed using HMMcopy. Results: CTCs (EpCAM+/cytokeratin+) were detected by CellSearch in 1/10 of patients (15 CTCs/7.5 ml). In CellSearch negative patients, cytokeratin+/vimentin- CTCs were detected in 1/3 patients analysed by Parsortix (6 CTCs/7.5 ml), and a large mesenchymal (cytokeratin-/vimentin+) circulating tumour microembolus was detected in 1/3 patients (comprising >50 CTCs). cfDNA was isolated from 10/10 patients (range 3 - 63 ng/ml of plasma, median 9.22 ng/ml). CNA analysis identified chromosomal losses and gains consistent with tumour origin. In 1/10 patients, analysis of cfDNA collected following disease progression on a clinical trial targeting DNA damage response pathways identified focal amplification at chromosome 7q including the gene CDK6. The clinical relevance of CDK6 amplification as a biomarker predicting response to the CDK4/6 inhibitor LEE011 is currently under clinical evaluation in the SIGNATURE study (NCT02187783). Conclusions: This is the first report of CTC and cfDNA detection in ACC, showing their combined analysis has potential as a liquid biopsy for tumour profiling to detect clinically actionable mutations. Liquid biopsies may also be a valuable tool for disease monitoring for early detection of tumour recurrence, which is a characteristic of this disease. As tumour profiling is further incorprated into treatment pathways, there is an urgent need for more large multicentre biomarker-led basket studies to test drug-biomarker combinations to develop new therapies and deliver patient benefit from these analyses. Legal entity responsible for the study: The Christie NHS FT. Funding: The ACC Research Foundation, CRUK Manchester Institute, and University of Manchester. Disclosure: All authors have declared no conflicts of interest.