Abstract
Circulating tumour DNA (ctDNA) are small fragments of DNA released by tumours
into the circulation. These fragments contain pathogenic mutations which can be
detected and quantified by various next generation sequencing approaches to monitor
tumour burden. Many ctDNA analyses rely on sequencing of tumour tissue using large
sequencing panels, querying hundreds of genes to find candidate ctDNA biomarkers for
follow-up. Whilst this approach has been successfully implemented to track changes in
tumour burden over time, it requires the sequencing of tumour tissue – which is
difficult to obtain and may not accurately represent the entire genomic landscape of
tumours. Hence, this study designed a targeted DNA sequencing panel to detect
pathogenic mutations directly from the plasma of gastric and colorectal cancer patients.
In addition, the criteria of selecting ctDNA biomarkers was investigated in colorectal
cancers by comparing the ability of mutations in tumour suppressor genes and
oncogenes to track changes in tumour burden throughout chemotherapy treatment.
The gastrointestinal cancer sequencing panel (GI cancer panel) designed in this study
successfully identified pathogenic mutations in the plasma of gastric and colorectal
cancer patients. By restricting the panel to only 21 genes, and designing specialised
oligonucleotide sequencing primers, a high analytical sensitivity was reached.
Mutations were successfully identified down to a frequency of 0.5%. This study also
identified technical challenges associated with ctDNA sequencing approaches –such as
distinguishing pathogenic mutations from sequencing errors and benign variants found
in the plasma. Finally, this study highlighted several factors that should be considered
when selecting ctDNA biomarkers – including the necessity to monitor multiple
mutations to better represent the total tumour burden. Overall, this study has generated
evidence supporting the implementation of ctDNA technologies into the healthcare
system to better manage gastrointestinal cancers.