Introduction: For early stage NSCLC, surgery with curative intent is the most common therapeutic option; however, tumour recurrence occurs in approximately 50% of cases and most commonly at distant sites. Analysis of CTCs represents a potential means of tracking cancer cells from the primary tumour and has the potential to provide insights into the metastatic process and mechanisms of resistance to therapy linking cell phenotype and genotype. Within the TRACERx study, we are carrying out whole genome analysis of single CTCs obtained from NSCLC patients undergoing surgery in order to examine the relationship between CTCs, the primary tumour and metastatic relapse.
Methods: Blood from 163 patients was collected at surgery from the tumour draining pulmonary vein and from peripheral vein in longitudinal samples. CTCs were enriched based on their EpCAM expression using CellSearch® and then enumerated according to their positivity for epithelial markers (Cytokeratins) and lack of detectable blood markers (CD45). Samples containing at least five CellSearch® CTCs (CS-CTCs) were processed through DEPArray™ for single cell isolation followed by whole genome amplification (WGA) using the Ampli1™ WGA kit. Genome wide copy number alterations (CNA) were detected through low-depth whole genome sequencing (WGS) and mutational analysis of individual CTCs was carried out using whole exome sequencing (WES). CTC genomic profiles were compared to the corresponding spatially separated tumour sections obtained at surgery.
Results: In a pilot study of 30 patients undergoing tumour resection, the presence of pulmonary vein CS-CTCs has been linked to poor clinical outcome (Crosbie et al, J Thorac Oncol, 2016). Here we extended the evaluation of pulmonary vein CS-CTCs to a further 122 patients and confirmed the initial observation that the presence of pulmonary vein CS-CTCs is a poor prognostic indicator. CNA analysis of 100 single CTCs from the first 12 patients revealed heterogeneous patterns between patients and within individual patients. Three types of pulmonary CS-CTC candidates were observed: Type 1, which share clear copy number changes with both tumour and other CS-CTCs; Type 2, where copy number changes were detected but were not shared with the tumour; Type 3, where no copy number changes were detected. Using WES of both CTCs and matching excised tumour regions, we identified common genetic alterations shared by CTCs and the tumour, as well as private genetic changes detected only in CTCs. We are currently evaluating metastatic biopsies from relapsing patients and comparing their molecular profiles to those seen in CS-CTCs obtained at surgery.
Conclusion: The combined tumour and CTC data show that single CTC analysis provides an additional layer of complexity with a valuable new perspective on tumour heterogeneity and early dissemination in NSCLC.
Citation Format: Francesca Chemi, Sakshi Gulati, Dominic G. Rothwell, Debbie Burt, Daniel Slan-Tan, Barbara Mesquita, Chris Wirth, Gareth Wilson, Jackie Pierce, Ged Brady, Charles Swanton, Caroline Dive. Single-cell molecular profiling of circulating tumor cells (CTCs) within the TRACERx study reveals heterogeneous patterns in early non-small cell lung cancer (NSCLC) [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 5601.