Abstract 1722: Novel multiplex liquid biopsy detection of ALK and ROS1 rearrangements and intratumoral heterogeneity in lung cancer using circulating tumor cells (CTCs) isolated from NSCLC patient bloods and TR-CytoTrapNanoTM technology
Authors: lee sangjun 
Published: July 2017

Introduction: ALK and ROS1 rearrangements are common gene alterations found in subsets of patients with NSCLC. Identification of ALK and ROS1 rearrangement in NSCLC is critical for highly active targeted therapies aiming for these two main tumorigenic drivers. Due to the similarity of tyrosine kinase domains between ALK and ROS1, many ALK inhibitors are also functional in treatment of ROS1 subtype NSCLC. For example, crizotinib, which is a standard therapy for advanced ALK-rearranged NSCLC is highly effective in advanced ROS1-rearranged genotype NSCLC patients. The mutational analysis of these genes requires serial tumor biopsying from primary lesions, which requires high risk clinical procedures. Further, the current tissue-based diagnostics, such as fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) are known to be challenging technically with limited sensitivity, subjective interpretation and inaccuracy in monitoring the current tumor status. The TR-CytoTrapNanoTM CTC technology is a blood-based assay to utilize CTCs as a biosource for mRNA extraction to detect genetic alteration or gene expression profiles including ALK and ROS1 gene rearrangements. In this study, we determined the variant specific expression of ALK fusion transcripts (EML4-ALK, KIF-ALK, and TGF-ALK) and ROS1 fusion transcripts (EZR-ROS1, SDC4-ROS1, TPM3-ROS1, LRIG3-ROS1, GOPC-ROS1, CD74-ROS1, and SLC34A2-ROS1) using multiplex digital droplet polymerase chain reaction(ddPCR) technology.

Method: The Peripheral Blood Mononuclear Cells isolated from NSCLC patient bloods run through the TR-CytoTrapNanoTM microfluidic system. The CTCs are enriched and captured on the chip surface conjugated with Anti-EpCAM targeting antibody, which specifically binds to CTCs of epithelial origin. Following lysis step facilitate total RNA release from trapped CTCs and then pure mRNAs are isolated using oligo-dT conjugated magnetic beads. The mRNAs are used for following ALK and ROS1 specific reverse transcription reaction to make cDNAs for either ALK or ROS1 fusion transcripts. The specific subtyping of ALK and ROS1 rearrangement are then validated using ddPCR.

Results and Conclusion: We tested several liquid biopsies from ALK and ROS1 rearrangement positive patients and corresponding positive data to FISH assays using solid tumor biopsies. Interestingly, some of the CTCs harboring ALK and ROS1 rearrangement has shown high intratumor heterogeneity in the subtypes of fusion partners. we envision that our novel TR-CytoTrapNanoTM CTC ALK/ROS1 rearrangement Assay to monitor treatment response and recurrence in NSCLC patients will be used to monitor treatment response and disease recurrence following treatment with Tyrosine Kinase Inhibitors (TKIs).

Note: This abstract was not presented at the meeting.

Citation Format: sangjun lee. Novel multiplex liquid biopsy detection of ALK and ROS1 rearrangements and intratumoral heterogeneity in lung cancer using circulating tumor cells (CTCs) isolated from NSCLC patient bloods and TR-CytoTrapNanoTM technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1722. doi:10.1158/1538-7445.AM2017-1722