Abstract C148: Rapid blood-based test for ALK, RET and ROS1 mRNA fusions and somatic variants in NSCLC
Authors: Mellert Hestia , Koch Kristina , Campbell Shannon , Pestano Gary 
Published: May 2016

Abstract
Tissue based methods for the detection of ALK fusions include FISH and IHC. Patients with advanced non-small cell lung cancer (NSCLC) may not be good candidates for biopsies and in cases where tissue is obtained, it is not always sufficient for testing. Additionally, determination of the full range of tumor heterogeneity and any impact of molecular variants on therapeutic responses may not be fully captured by the current FISH and IHC tests being conducted for ALK. For these reasons, blood-based testing that uses PCR and NGS methods for detection of mRNA fusion transcripts and somatic variants in circulation are becoming more clinically relevant. Pre-analytic as well as primer design are critical parameters in the development of circulating RNA tests. We have approached the pre-analytic variables by assessing multiple potential sources of fusion RNAs in prospectively-collected plasma samples, including cell-free RNA (cfRNA) and exosomal RNA. Additionally, test development for ALK, RET and ROS1 (ARR) utilized fusion-partner agnostic primers, only requiring knowledge of the fusion kinase sequence. This test technology has previously been shown in FFPE-extracted mRNA specimens to have the unique capability to identify common fusions, including EML4-ALK variants 1 and 3a/b, as well as rare fusions and novel transcript variants. In this study we have extended the capability of this technology to plasma samples from donors previously diagnosed with NSCLC. Data generated during the development of this test involved extraction of RNA from cell lines and donor plasma, processing to cDNA, adaptor ligation and two-step anchored PCR with specificity for human ALK, RET and ROS1. Amplicons were sequenced on a MiSeq (v2 chemistry) and reads were mapped to the human genome and a fusion specific database in order to identify the specific fusion partner(s). Test turn-around time from RNA extraction to results averaged 72 hours. With this test system we evaluated known variant-positive cell lines for EML4-ALK, SLC34A2-ROS1 and CCDC6-RET, as well as remnant prospectively-collected plasma from NSCLC donors (females of less than 65 years of age, and wild-type for circulating EGFR sensitizing and KRAS somatic variants). All cell lines were positive for the respective fusions (lower limit of input RNA at 25ng) and negative in test cross-reactivity experiments (up to 250ng input RNA). The presence of the fusions in the cell lines were independently confirmed with variant-specific qPCR assays. We additionally tested cell-line spiked human donor samples and successfully demonstrated detection of cell-line derived ARR fusions and donor-derived wild-type ALK, RET & ROS1. All human donor samples passed QC measures based on control gene expression. Ongoing experiments are being conducted to further develop this test and evaluate its relevance for routine blood-based clinical laboratory testing using plasma from patients diagnosed with NSCLC.
Citation Format: Hestia Mellert, Kristina Koch, Shannon Campbell, Gary Pestano. Rapid blood-based test for ALK, RET and ROS1 mRNA fusions and somatic variants in NSCLC. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C148.