Abstract
Circulating tumour DNA (ctDNA) refers to short extracellular DNA fragments (~166 bp) released by tumour cells as a result of apoptosis, necrosis, or active secretion. Due to its short half-life (16 mins – 2.5 hours), ctDNA can be detected in the bloodstream to provide reliable and real-time representation of the disease, particularly when tissue biopsies are difficult to obtain, or the primary tumour location is unknown. This study explored and tested various methods for the early detection and improved diagnosis of cancer through ctDNA analysis. Specifically, three research projects were investigated: (1) serial monitoring of breast cancer patients through mutation-based ctDNA detection, (2) analysis of cell free DNA (cfDNA) fragmentation profiles in gastric cancer, and (3) developing a novel indwelling device for improved ctDNA capture.
Firstly, this study assessed the utility of targeted next-generation sequencing (NGS) and droplet digital PCR (ddPCR) for the serial detection of ctDNA in breast cancer patients undergoing systemic therapy in New Zealand. Two patient groups were examined — one involving 35 patients with metastatic breast cancer and another involving four patients with early to late-stage breast cancer. Findings indicated that ddPCR-mediated serial ctDNA surveillance was highly sensitive and specific, outperforming the conventional CA15-3 biomarkers and providing a valuable adjunct to CT scans for monitoring treatment response and detecting disease progression. This minimally invasive approach offered real-time insights into the mutation dynamics and drug resistance, accurately reflecting both primary and metastatic tumour burden without the reliance on tissue biopsies. These results validated the potential utility of ctDNA for routine clinical management of breast cancer patients across various tumour stages, particularly in settings where access to imaging modalities is limited.
Secondly, this study evaluated the genome-wide fragmentation profiles of the pre- and post- surgery cfDNA from six gastric cancer patients compared to healthy individuals, using a modified version of DELFI (DNA evaluation of fragments for early interception) and various bioinformatics analyses. This study provided a detailed genomic analysis of promoter fragment patterns, transcription factor motifs, and chromosomal copy number alterations (CNAs) that are enriched in the blood of gastric cancer patients. These findings underscored the potential of cfDNA fragmentation as a minimally invasive tool for diagnosing gastric cancer in patients with low tumour burden.
Lastly, this study investigated different methods for developing a novel indwelling device for improved ctDNA capture. Various 3D biofabrication techniques (electrospinning, melt electrowriting) and surface functionalisation methods (ARGET ATRP grafting, GraftFastᵀᴹ, click chemistry reaction) were explored for the attachment of bioactive compounds onto different substrate materials (polymers, stainless steel) to enhance the overall capture efficiency and blood biocompatibility of the device. This study demonstrated promising methodologies for ctDNA capture while also addressing ongoing challenges in achieving blood biocompatibility and reproducibility. Overall, these findings lay the foundation for future innovations in advancing biomedical device technologies for ctDNA-based cancer research.
Collectively, these findings contribute valuable insights into the evolving landscape of cancer detection and surveillance, underscoring the importance of integrating ctDNA technologies into routine clinical practice for more accurate diagnosis, continuous treatment monitoring, and the development of personalised therapeutic strategies to improve the clinical management of cancer patients.