Abstract B36: Identifying single-cell gene expression and EGFR mutation profile heterogeneity in NSCLC patients’ CTCs
Authors: Owen Sarah , Lo Ting-Wen , Fouladdel Shamileh , Zeinali Mina , Keller Evan , Azizi Ebrahim , Ramnath Nithya , Nagrath Sunitha 
Published: June 2020

The efficacy of targeted therapies was first shown in patients with non-small cell lung cancer (NSCLC) who possess activating epidermal growth factor receptor (EGFR) mutations that responded to tyrosine kinase inhibitors (TKIs). Current standard of care is to screen metastatic patients for the presence of EGFR mutations to determine if they qualify for TKI treatment. Due to the invasiveness of a tumor biopsy, this characterization is typically only done at the time of diagnosis. This single time-point determines the entire treatment plan. Despite strong initial response to TKI therapy, most patients develop resistance within months, most commonly by acquiring a secondary EGFR mutation. Identifying resistance through current monitoring techniques, such as measuring tumor volume, can lead to a delay in detection. Alternatively, circulating tumor cells (CTCs) are cancer cells present in the blood that provide access to tumor cells without the requirement of a biopsy. Shed from the tumor into the vasculature, CTCs circulate throughout the body before a fraction extravasate, leading to metastatic sites. CTCs have been shown to carry tumor-matched characteristics in both genotype and phenotype. Due to their ease of access, they can be used to serially track patients’ conditions to detect the early emergence of new tumor clones and, potentially, therapeutic resistance. One of the major limitations of CTCs’ clinical utility is their low abundance in the blood. Our group previously developed the Labyrinth, a high-throughput, label-free microfluidic technology, which enables rapid and efficient CTC enrichment. These CTCs are used for enumeration or single-cell transcriptomic analysis. Initial single-cell analysis was performed through highly multiplexed RT-qPCR that compared 96-gene expression profiles to unravel inter- and intrapatient CTC heterogeneity. We recently developed a complimentary single-cell analysis approach using digital PCR (dPCR) to detect the presence of sensitizing EGFR mutations, exon 19 deletion and L858R, and resistance mutation, T790M. dPCR is an ultrasensitive approach with single-molecule resolution, which enables detection and quantification at a single-cell level by partitioning the sample into individual PCR reaction droplets. This resolution can distinguish if a cell is homo- or heterozygous for a mutation and each allele’s relative expression level. We validated this system for single-cell analysis using lung cancer cell lines and demonstrated its agreement with qPCR results. We present initial findings of EGFR mutation screening in CTCs from a small cohort of NSCLC patients with known EGFR status. We highlight the single-cell heterogeneity of CTCs within a patient at both the gene expression and EGFR mutation level. By tracking these patients’ CTCs over time, we may be able to identify the emergence of resistant clones sooner than can be done in the clinic. This may allow for more rapid treatment modification and lead to improved patient outcomes.

Citation Format: Sarah Owen, Ting-Wen Lo, Shamileh Fouladdel, Mina Zeinali, Evan Keller, Ebrahim Azizi, Nithya Ramnath, Sunitha Nagrath. Identifying single-cell gene expression and EGFR mutation profile heterogeneity in NSCLC patients’ CTCs [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B36.