Detection of somatic mutations and copy number alterations in circulating tumour DNA in patients with primary breast cancer by next-generation sequencing: an in-depth analysis of the Neocent trial to evaluate the efficacy of neoadjuvant therapy

Breast cancer is one of the most common malignant tumours in women worldwide. With the continuous advancement of new technologies, more accurate and earlier detection and diagnosis of cancer have become possible. This thesis explored the use circulating tumour DNA (ctDNA) in patients with breast cancer. In the Neocent study, paired tumour whole exome sequence data was available before and after 21 weeks neoadjuvant therapy and bioinformatics approaches were used to compare exome profiles. Shallow whole genome sequencing (sWGS) was also used to explore somatic copy number alterations (SCNA) in ctDNA from in these patients. A related study in patients with metastatic breast cancer (MBC) evaluated NGS analysis of specific mutations using the Oncomine breast cancer cfDNA assay and results were compared with detection of SCNA by sWGS. 

Results from two bioinformatics workflows, Sequenza and SuperFreq, In Neocent tumour exome data showed that SuperFreq analysis was more reflective of focal gene-specific amplification than Sequenza analysis as determined by ddPCR analysis. The results of sWGS showed that unfortunately Neocent baseline plasma had limited or no detectable ctDNA at diagnosis.

In the MBC cohort (the CTCM cohort study), results showed that ReproSeq was more sensitive than Oncomine in revealing SCNA. However, Oncomine was more sensitive in detecting ctDNA overall. Finally, in a pilot study comparison of Oncomine ctDNA profiles and treatment outcomes in the ALERT cohort demonstrated the utility of ctDNA testing in monitoring treatment response. 

In conclusion, results presented in this thesis demonstrate the potential of ctDNA analysis to monitor breast cancer patients at the time of neoadjuvant therapy and in metastatic disease to inform clinical decision-making.