Genomic Profiling and Characteristics of Circulating Tumour Cells in Patients with Small Cell Lung Cancer

Abstract

Genomic Profiling and Characteristics of Circulating Tumour Cells in Patients with Small Cell Lung Cancer.A Thesis submitted to the University of Manchester for the Degree of PhD by Louise Rosalyn Carter, May 2015.Background Small cell lung cancer (SCLC) is an aggressive, highly metastatic disease with dismal prognosis. Chemoresistance, both intrinsic and acquired, represents one of the major challenges in the management of SCLC contributing to the poor outcomes seen. Response rates to first-line chemotherapy are high, but the responses are not durable with short progression-free survival and significantly reduced response rates to further treatment seen. Patients with chemorefractory disease have particularly poor survival, even when compared to SCLC as a whole. Biopsies, particularly serial biopsies, are challenging to obtain in SCLC for research. Circulating tumour cells (CTCs) are prevalent in SCLC and represent a potential minimally invasive alternative source of tumour material for molecular analysis. The ambitious aim of this thesis was to develop methods for the molecular analysis of CTCs in SCLC, to interrogate the genomic landscape of this disease and to explore mechanisms of resistance to chemotherapy.Methods To monitor tumour genetics in SCLC, a CTC workflow was developed using the CellSearch system for CTC enrichment followed by DEPArray CTC isolation. Using this workflow, CTCs were isolated from chemorefractory patients and chemoresponsive patients' blood samples, prior to chemotherapy and again at progression with relapsed disease. Following single-cell whole genome amplification, Sanger Sequencing, TAm-Seq, low coverage whole genome sequencing and whole exome sequencing (WES) of CTCs and CTC-derived explants (CDX) were used to investigate mutations and to generate genome-wide patterns of copy number alterations (CNA).Results Hallmark SCLC molecular abnormalities such as TP53 mutations and copy number loss in tumour suppressor genes such as RB1 (previously identified in bulk tumour profiling), were noted in the isolated SCLC CTCs and in the patient matched CDX models in mice developed by colleagues in our group. Distinct CNA profiles were found in the CTCs isolated from patients with chemorefractory disease compared to those isolated from patients with chemoresponsive disease. A potential signature of 760 genes with statistically significant change in copy number between the two groups of patients' CTCs was identified. This signature of CNA changes were not seen in CTCs isolated when initially chemoresponsive patients relapsed when compared to the baseline samples, however new mutations were identified by WES between presentation and relapse.Conclusion SCLC CTCs, the invasive subset of tumour cells, reflect and share the common mutational changes identified in bulk SCLC tumour sequencing. The identification of a signature of CNAs potentially associated with intrinsic resistance, yet none associated with the development of acquired resistance, suggests that there may be different mechanisms underlying these two processes. The potential research utility of CTCs in SCLC has been confirmed with the results of this thesis, which has opened up new avenues to study acquired and intrinsic chemoresistance, as well as a route to identify much needed new drug targets.

Date of Award	31 Dec 2015
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Caroline Dive (Supervisor) & Gerard Brady (Supervisor)