Use of liquid biopsies as prognostic and diagnostic marker for non-small cell lung cancer

This thesis was submitted for the award of Doctor of Philosophy and was awarded by Brunel University LondonLung cancer survival remains poor in the western world due to late presentation in most cases, leading to difficulty of treatment in these advanced and metastatic patients. Therefore, the development of robust biomarkers for prognosis and monitoring treatment response and relapse would be of great benefit. Blood based biomarkers, referred to as ‘liquid biopsies’ are becoming increasingly popular in the cancer field, due to their non-invasive nature compared to conventional biopsies. Circulating tumour cells (CTCs) are cells that have detached from the primary tumour, enter the blood stream and can be of diagnostic or prognostic value in several cancers. Similarly, circulating free tumour DNA (ctDNA), are fragments of tumour derived DNA, that can be detected in plasma. In this study, we used liquid biopsies to identify novel biomarkers of prognostic or diagnostic value, by characterising and enumerating of CTCs, measuring of circulating cell-free DNA (ccfDNA) and inflammatory biomarkers and identification of differentially expressed genes in vitro, following silencing of a key long non-coding RNA. Following series of validation experiments using lung cancer cell lines (A549 and H1975), we have characterised CTCs from NSCLC patients using multi-spectral imaging flow cytometry (ImageStream) against multiple targets such as EpCAM, PanCK, TTF-1, CK7 and CD45 as a marker to exclude white blood cells. We have investigated the impact of surgically induced trauma using peripheral blood from pre- and post-operative non-small cell lung cancer (NSCLC) patients undergoing thoracotomy or video-assisted thoracoscopic surgery (VATS) resection. CTCs were increased in post-operative blood samples in 54 NSCLC patients. Patients that underwent thoracotomy instead of VATS had higher numbers of EpCAM (p=0.004) and PanCK-labelled (p=0.03) CTCs post-operatively (statistical test; paired t-test). ccfDNA and DNA integrity index were also significantly increased in post-operative samples (p=0.0009 and p=0.04), with concomitant increase of IL-6 and IL-10 levels in the same cohorts (p=0.0004 and p=0.034 respectively). In this part of the thesis we have shown the potential clinical utility of several biomarkers from liquid biopsies to guide peri-operative management. This is of particular importance since the long-term goals of lung cancer surgery include improved length and quality of life, and surgical intervention is not always an option. Finally, based on previous studies from laboratory, the lncRNA X-inactive specific transcript (XIST) appeared to be of diagnostic value. Here, we have made use of siRNA to silence this gene in two lung cancer cell lines (A549 and H1975) and RNA-Seq to identify differentially expressed genes (DEGs). We have found that these genes are involved in signal transduction, energy and metabolic pathways, and cell communication. Moreover, we expanded our observations making use of in-silico tools, demonstrating an association of XIST expression associated with TSIX, hnRNPu, Bcl-2, and BRCA1. This cluster of genes collectively, has a strong discriminatory power between controls and lung cancer, implying a diagnostic potential. The holistic approach into the interrogation of multiple readouts using liquid biopsies, indicated a potential clinical utility of several biomarkers from liquid biopsies to monitor disease burden peri-operatively includes more accurate prognostication for patients as well as guiding the role of adjuvant therapy (chemotherapy and radiotherapy). Moreover, it paves a research path for further studies into the role on lncRNAs in this particular malignancy