Abstract 5581: Clinical validation of a highly sensitive assay to detect mutations in plasma from advanced lung adenocarcinoma patients
Authors: Wikman Harriet , Mohme Malte , Janning Melanie , Sartori Alexander , Lamszus Katrin , Riethdorf Sabine , Irwin Darryl , Westphal Manfred , Loges Sonja , Pantel Klaus 
Published: August 2018

Abstract
The use of different liquid biopsy tools including circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs) have shown great promise for the use in personalized cancer diagnostics, treatment monitoring, and research, especially among metastatic patients. The power of ctDNA analyses in early detection of acquired resistance against e.g. EGFR-targeted therapies in non-small cell lung cancer (NSCLC) have been demonstrated in several studies. However, NSCLC patients display lower ctDNA and CTC concentrations than expected and in particular brain metastases pose a challenge, probably due to the blood-brain barrier. In this study, we used a novel highly sensitive assay to detect clinically relevant mutations in ctDNA and detected CTCs in blood from metastatic NSCLC patients. 56 metastatic NSCLC cases were enrolled in this study. 37 patients presented with brain metastases and 20 of them had brain as the only site of metastasis (oligo-metastatic disease). Cell free (cf)DNA was isolated from 1.5-2ml of plasma using the Circulating Nucleic Acid kit (Qiagen). The ctDNA quantity was measured by Qubit Fluorometer (Thermo Fisher) and the quality was assessed by Tapestation (Agilent technologies). The UltraSEEK™ Lung Panel on the MassARRAY® System (Agena Bioscience) was used for the detection of mutations in five genes EGFR, KRAS, BRAF, ERBB2 and PIK3CA covering 67 different hot spot mutation loci. CTCs were detected by the CellSearch system. The total cfDNA concentration yields were low in most cases (median; 21ng/30µl, mean; 39ng/30µl, ± SD 2.6 ng/µl isolated from 1.5-2ml of plasma). Mutations could be detected in 26 samples (46.4%), with the variant allele frequency ranging between 0.1% and 5%. EGFR mutations were detected in 23.2% (13/56), KRAS in 21.4%, ERBB2 in 7.1%, PIK3CA in 3.6% and BRAF in 1.8% of the samples. Mutations were detected in 57.9% of patients with peripheral (non-brain) metastases, whereas 40.5% of brain metastatic patients had mutations detected in blood. Among oligo-metastatic patients, 30.5% had detectable mutations in plasma. In 42 samples, the EGFR mutation status in the primary tumor tissue was also determined. In 90.5% of samples, concordant results between plasma and tissue were found. In two patients, an EGFR mutation was only detected in the plasma, whereas in two other samples the mutation was only detected in the tumor tissue. In 46 patients, CTCs were measured. In 11 patients (23.9%), ≥ 1 CTC was detected. In 64.7% of patients, an informative result was obtained when results from both liquid biopsy approaches were combined. The UltraSEEK technology was able to detect mutations in cfDNA in plasma from NSCLC patients with limited, oligo-metastatic tumor burden, low amounts of total cfDNA, and very low fractions of ctDNA. Parallel assessment of CTCs in patients without detectable ctDNA enlarged the population of patients amenable to successful liquid biopsy analysis.
Citation Format: Harriet Wikman, Malte Mohme, Melanie Janning, Alexander Sartori, Katrin Lamszus, Sabine Riethdorf, Darryl Irwin, Manfred Westphal, Sonja Loges, Klaus Pantel. Clinical validation of a highly sensitive assay to detect mutations in plasma from advanced lung adenocarcinoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5581.