Establishing methods for analysis of DNA methylation in breast cancer and cell-free circulating DNA

Abstract

Breast cancer is the most common cancer in women worldwide. To date, diagnosis and metastasis monitoring are mainly carried out through tissue biopsy, a very invasive procedure limited only to certain locations and not always feasible in clinical practice. Tumour cells release DNA into the blood as circulating cell-­free DNA (cfDNA), which can be sampled from circulating blood, an approach known as liquid biopsy. This provides a resource for biomarkers that could allow the use of minimally invasive liquid biopsies for cancer-­related research, diagnostics, prognostics, and targeted therapy. The levels of cfDNA have already been shown to be higher in cancer individuals than healthy individuals, and correlate with tumour metastasis, response to therapy and recurrence. Recent technological advances have enabled the identification of both genetic and epigenetic aberrations in cfDNA that reflect changes also found in patients’ tumours. The host group performed methylation analysis using the Illumina EPIC Methylation Array, which interrogated CpG dinucleotide methylation at over 850,000 DNA sites. A total of 3172 CpGs showed median methylation differences of more than 40% between tumour and buffy coat of patients with breast cancer. This MRes project aimed to establish methods for detecting these methylation changes between matched tumour samples and leucocytes of breast cancer patients, and in cfDNA by using the Fluidigm 48.48 Access Array microfluidics system and the Illumina MiSeq sequencer. This approach provided quantitative, medium-­throughput targeted measurement of DNA methylation at single nucleotide resolution. Finally, bisulfite pyrosequencing was used as a sensitive validation technique for detecting differences in CpG methylation, providing a set of potential biomarkers that could be reliably detected by circulating tumour DNA-­based tests. Translating the alterations that are seen in the primary tumour into an assay that is applicable to cfDNA will have important diagnostic implications, such as monitoring tumour progression, drug response and disease recurrence, as well as the early detection of cancer, which could ultimately complement or even avoid the need for tumour tissue biopsies.