Title

Assessment of circulating tumour cells in lung cancer patients

Author Identifiers

Emmanuel Acheampong

https://orcid.org/0000-0002-5338-3258

Date of Award

2022

Degree Type

Thesis

Degree Name

Doctor of Philosophy

School

School of Medical and Health Sciences

First Advisor

Elin S. Gray

Second Advisor

Michael Millward

Third Advisor

Weitao Lin

Fourth Advisor

Aaron Beasley

Abstract

Lung cancer is among the most prevalent forms of cancer and remains the leading cause of cancer-associated deaths globally. Traditionally, lung cancers are classified as either non-small cell lung cancer (NSCLC) (85%) or small cell lung cancer (SCLC) (15%). About 60% of all cases are diagnosed at an advanced stage, at which the 5-year survival is only 4%. Anti-programmed cell death-1 and its ligand 1 (anti-PD-1/PD-L1) therapies have significantly improved the outcomes for lung cancer patients in recent years. However, prognosis and understanding of an individual patient’s lung cancer are often limited by tumour accessibility. Tissue biopsies are invasive, costly, and technically challenging procedures, posing risks to the patient. Circulating tumour cells (CTCs) are very attractive tumour surrogates that could serve as “liquid biopsy” with the advantage to be a low–to–null invasive and real-time approach compared to conventional tissue biopsies. Increasing evidence suggests that CTCs counts can serve as a prognostic biomarker for lung cancers. Notably, phenotypic, and molecular characterisation of CTCs may offer important clinical information for guiding personalised medicine.

The studies in this dissertation assessed the potential of CTCs to provide information that could aid the management of lung cancer patients. We carried out a series of investigations covering a systematic review and meta-analysis of programmed cell death ligand-1 (PD-L1) expression on tumour samples and CTCs, a methodological study to improve phenotypic characterisation of CTCs for PD-L1 expression and its application in the clinical settings, and a study using singlecell genomics to uncover novel subpopulations of CTCs.

The first chapter of the thesis includes an introduction to lung cancer and a thorough review of the literature on immunotherapy in lung cancer as well as CTCs. Chapter 2 describes a comprehensive review and meta-analysis of PD-L1 expression on tumour cells in SCLC from 27 studies enrolling a total of 27,292 patients. Our results revealed that the prevalence of PD-L1 expression in SCLC tumour cells was heterogeneous across studies. This heterogeneity was significantly moderated by factors such as cut-off values used for scoring PD-L1 staining by immunohistochemistry, and assessment of PD-L1 staining patterns as membranous and/or cytoplasmic. Following these findings, Chapter 3 covers a study carried out to address the feasibility to quantify PD-L1 expression on CTCs in SCLC patients. We develop an EpCAM targeting magnetic bead-based CTC isolation method as a surrogate for the CellSearch method, as this is the gold standard for CTC enumeration and the most used SCLC CTC isolation platform in the clinical setting. Using our immunomagnetic isolation technique, we compared detection rates of CTCs to those isolated using the microfluidic CTC enrichment device - Parsortix system, which separates cells by size exclusion. Detected CTCs were used to assess PD-L1 expression. We identified a subpopulation of EpCAM-negative SCLC CTCs, indicating that epitope-independent methods can detect additional CTCs missed by EpCAM basedcapture. The study also demonstrated that PD-L1 expression can be quantified on CTCs detected in SCLC patients.

In parallel, we questioned whether blood is the alternative for PD-L1 expression in NSCLC patients based on several published studies that have assessed PD-L1 expression on CTCs in NSCLC patients. The review in Chapter 4 indicates that the analysis of PD-L1 on CTCs is feasible and PD-L1 expression could be detected before and after first-line therapy. However, there was limited evidence of whether PD-L1 expression on CTCs could predict response to anti-PD-1/PDL1 treatment.

Chapter 5 describes a study in NSCLC patients to improve the detection of relevant CTC phenotypes and interrogate them for PD-L1 expression. We simultaneously identified circulating cells with epithelial origin and cells with mesenchymal features in patients with NSCLC by combining the Parsortix system with a modified sequential fluorescent quenching and restaining protocol. Nevertheless, none of the detected circulating cells expressed PD-L1 protein. Furthermore, a subset of mesenchymal-featured cells was confirmed as cancer cells via whole genome amplification (WGA) and low-pass whole-genome sequencing (LP-WGS) which revealed copy number alterations (CNAs) in several genomic regions.

Lastly, the general discussion underscores how specific CTCs enrichment techniques are required for lung cancers according to their phenotypic characteristics. The results question the potential of CTCs for evaluating PD-L1 expression and the need for systematic clinical validation. Finally, the prospect of CTC genomic analysis is highlighted as it provides an opportunity to timely recognise patients harbouring deleterious alteration and new treatment targets. We conclude by proposing future directions building upon the findings presented in this thesis.

Access Note

Access to this thesis is embargoed until 5 July 2024.

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