Abstract 407: Gene expression and mutation profiles of single lung circulating tumor cells
Authors: Owen Sarah , Lo Ting-Wen , Fouladdel Shamileh , Azizi Ebrahim , Ramnath Nithya , Nagrath Sunitha 
Published: August 2019

In the era of personalized medicine, many targeted therapies have been developed against cancer-specific antigens, which have greatly improved patient outcome. The efficacy of targeted therapies was first shown in patients with non-small cell lung cancer (NSCLC) containing activating epidermal growth factor receptor (EGFR) mutations who responded to tyrosine kinase inhibitors (TKIs). To determine if a patient qualifies for targeted therapy, the patient must undergo a tumor biopsy. Usually done at the time of diagnosis, this single time-point examination dictates the treatment plan. Despite strong initial response to TKI therapy, nearly all patients develop resistance, most commonly by acquiring a secondary EGFR mutation. At the time of halted response, an additional biopsy may be necessary to screen for the presence of these secondary, resistance-causing mutations.

Circulating tumor cells (CTCs) are cancer cells present in the blood that provide an alternative to an invasive tumor biopsy. Shed from the primary tumor, CTCs circulate throughout the vasculature, and extravasate leading to metastasis. CTCs have been shown to carry tumor-matched characteristics in both genotype and phenotype. Easily accessed through a blood draw, they can be used to serially track patients throughout treatment to detect the early emergence of new tumor clones.

One of the biggest hurdles with CTCs being clinical prognostic markers is their low abundance in the blood. We have previously developed a high-throughput, label-free microfluidic technology, the Labyrinth, to isolate CTCs from the blood of breast, lung, and pancreatic cancer patients. This technology allows for quick and efficient isolation of CTCs from the millions of surrounding blood cells. After isolation, the CTCs are used for enumeration or single cell analysis. The single cell gene expression analysis performs a highly multiplexed RT-qPCR of a pre-designed 96 gene panel.

Here we report a complementary single cell analysis approach to detect the presence of EGFR point mutations, L858R and T790M, implicated in TKI response using digital PCR (dPCR). dPCR is a highly sensitive approach which enables detection and quantification a single-cell level. dPCR relies on small sample loading and partitions the sample into individual PCR reaction droplets. This single molecule resolution provides the ability to distinguish if a cell is homo- or heterozygous for a mutation as well as their relative expression levels. We have validated this system for single cell analysis using lung cancer cell lines with known L858R and T790M mutation status. We present initial findings of EGFR mutation screening of lung CTCs from patients with known EGFR status based on tissue profiling.

By tracking patients over time, comparing the CTC profiles, and correlating it to patient outcomes, we may be able to better identify early treatment resistant clones and adjust their treatment to improve patient outcome.

Citation Format: Sarah Owen, Ting-Wen Lo, Shamileh Fouladdel, Ebrahim Azizi, Nithya Ramnath, Sunitha Nagrath. Gene expression and mutation profiles of single lung circulating tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 407.