Molecular Profiling of Circulating Tumour Cells (CTCs) in Non-small cell lung cancer within the TRACERx study of Intratumoural Heterogeneity and Evolution


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Sakshi Gulati1,Francesca Chemi2,Dominic G Rothwell2,Deborah Burt2,Barbara Mesquita2,Christopher Wirth2,Gareth Wilson3,Jackie Pierce2,Ged Brady2,Charles Swanton4,Caroline Dive5
1Postdoctoral Scientist,2Cancer Research UK Manchester Institute,3The Francis Crick Institute,London, UK,4The Francis Crick Institute,London, UK and and on behalf of TRACERx consortium and Cancer Research UK Manchester/UCL Lung Cancer Centre of Excellence,5Cancer Research UK Manchester Institute and on behalf of TRACERx consortium and Cancer Research UK Manchester/UCL Lung Cancer Centre of Excellence

Abstract

Background

Circulating tumour cell (CTCs) profiling is gaining momentum as multiple studies demonstrate the potential of ‘liquid biopsy’ to study tumour biology. The TRACERx study (TRAcking non-small cell lung Cancer Evolution through therapy [Rx], ClinicalTrials.gov number, NCT01888601) is a prospective study in Stage I-III non-small cell lung cancer (NSCLC). Within this study, we are profiling pulmonary CTCs to establish their utility for detection of putative driver mutations and copy number aberrations (CNA) and determine whether number, heterogeneity or genetic status of CTCs isolated from blood just before surgical resection can predict tumour recurrence and aid subsequent therapy selection.

Method

CTCs were enriched, enumerated, isolated, biobanked and analysed using  our standard operating procedures. Next generation sequencing (NGS) used for both CNA and mutational profiling (whole exome sequencing WES) of single and pooled CTCs (and WBC controls). CTC genetic profiles are being compared to the genetic profiles of spatially separated resected tumour sections obtained at the same time as pulmonary blood sample used for CTC enrichment. In addition, metastatic biopsies and peripheral blood samples are being collected from relapsing patients to evaluate which CTC clones were responsible for metastasis.  Further, a pipeline to optimise variant calling from this dataset is under development.

Conclusion

We have established a robust workflow for enrichment, enumeration, isolation and molecular analysis of CTCs from early stage NSCLC patients in the TRACERx study which can be used to compare to matching tumour. Over 100 patient blood samples have been processed with CTCs isolated from 27 patients. NGS has been applied to >70 CTCs and WBC controls and initial CNA profiling and WES have been used to identify molecular changes shared by tumour and CTCs. On-going analysis will determine how the relationship between CTC and tumour genomic profiles shed light on NSCLC evolution, heterogeneity and recurrence.