PO-458 A multicenter study to assess EGFR mutational status in plasma: focus on an optimisedworkflow for liquid biopsy in a clinical setting

Abstract

IntroductionA multicenter study was performed in Belgium to establish an optimal workflow for liquid biopsy, consisting of circulating cell-free tumour DNA (ctDNA) analysis.Material and methodsEGFR ctDNA mutational analysis was performed on 549 plasma samples from 234 non-small cell lung cancer (NSCLC) patients. The influence of (pre-) analytical variables on concentration, allele frequency (AF) and sensitivity were investigated. These variables include centrifugation, transit time, temperature and the presence of brain (BM) and extra thoracic metastases (ETM).Results and discussionsSensitivity of ctDNA analysis is influenced by an interplay between increased plasma volume (p<0.001) and short transit time (p=0.018). Multistep, high-speed centrifugation both increases plasma generation (p<0.001), and reduces genomic DNA (gDNA) contamination. A longer transit time also increases the risk of hemolysis (p<0.001), which has a negative influence on sensitivity (p=0.047). Temperatures below 10°C were also shown to have a negative effect. The presence of ETM was found to be strongly associated with ctDNA detection (p<0.001), as well as AF (p=0.034). The concentration and AF in these patients were also significantly higher compared to patients in whom only BM were present. Furthermore, the original activating mutations were always detected in a higher concentration and AF than the T790M mutation (p=0.003, and p=0.002, respectively).ConclusionOptimisation of (pre-) analytical variables is key to successful ctDNA analysis. Sufficient plasma volumes without hemolysis or gDNA contamination can be achieved by using multistep, high speed centrifugation coupled with short transit time and precautions in case of low temperatures. Deviating pre-analytical variables, low levels of ctDNA in the circulation due to BM and no ETM, might hinder ctDNA detection. In case of using liquid biopsy as a tool to detect resistance, the detection of low AF of the original activating mutation without the T790M resistance mutation might be indicative of low ctDNA levels in the circulation. If clinically possible, a new liquid biopsy in a few weeks might reveal the T790M mutation. A tissue biopsy is advisable in case of either no detectable ctDNA or high AF of the original activating mutation alone. The detection of the T790M mutation in the ctDNA is sufficient to initiate osimertinib therapy. In this study, an optimised workflow for liquid biopsy has been established which might aid in a further standardisation and implementation in clinical practice.