Abstract 5577: Single cell mutation and gene expression co-analysis of lung circulating tumor cells
Authors: Owen Sarah , Lo Ting-Wen , Fouladdel Shamileh , Azizi Ebrahim , Nagrath Sunitha 
Published: August 2018

With the aim of personalized medicine, there have been many targeted therapies developed against cancer-specific antigens. The efficacy of targeted therapies was first shown in lung cancer patients containing activating epidermal growth factor receptor (EGFR) mutations who responded to tyrosine kinase inhibitors. Despite the promise of these EGFR targeted therapies, lung cancer patients routinely acquire an additional EGFR mutation, T790M, which causes treatment resistance. In order to determine if a patient qualifies for targeted therapy, they must get an invasive tumor biopsy, and may require a second biopsy if they stop responding to treatment. Circulating tumor cells (CTCs) are shed from the primary tumor into the bloodstream, a fraction of which lead to the development of metastases. CTCs can be isolated from a minimally-invasive blood draw, termed a “liquid-biopsy”, and their increased presence in the blood correlates with poor patient prognosis. CTCs present an approach to track and screen patients during treatment to determine if they acquire new mutations or other aggressive cell phenotypes. CTCs have been shown to be highly heterogeneous, existing in phenotypic states across the entire epithelial to mesenchymal transition spectrum as well as contain both primary tumor matched and unmatched mutations. We have previously established a workflow for the isolation and single cell characterization of CTCs. CTCs are isolated from the blood using a novel high-throughput inertial microfluidic device, the Labyrinth. This device has been optimized using cancer cell lines across many cancer types and has been used to process hundreds of blood samples from breast, lung, and pancreatic cancer patients for CTC enumeration. After CTC isolation using the Labyrinth, single cell suspensions are generated for lysis and RT-qPCR for up to 96 single cells using a 96-gene panel. Here we present initial results for the incorporation of mutation screening on the same single cells using digital PCR (dPCR). dPCR relies on small sample quantity to load one target molecule per reaction droplet for end-point PCR analysis. Due to its single molecule resolution, it has increased sensitivity compared to other traditional PCR methods, making it an ideal platform for single cell mutation screening. This enables us to determine if the cell is homo- or heterozygous, as well as quantify the relative expression in heterozygous mutations. We have validated the technology for single cell mutation detection of EGFR mutations, L858R and T790M, in addition to other genes such as TP53 and KRAS. The specificity of the EGFR mutation detection assays was confirmed using lung cancer cell line H1975, which is known to carry these two heterozygous mutations. Ultimately, the ability to couple single cell mutation and gene expression analysis using CTCs may allow oncologists to better monitor treatment response to guide the use and duration of targeted therapies.

Citation Format: Sarah Owen, Ting-Wen Lo, Shamileh Fouladdel, Ebrahim Azizi, Sunitha Nagrath. Single cell mutation and gene expression co-analysis of lung circulating tumor cells [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 5577.