Harnessing extrachromosomal circular DNA for liquid biopsy analysis

Background Liquid biopsy analysis refers to methods designed to detect tumour-specific material (e.g., circulating tumour cells or tumour DNA) in body fluids, such as urine or blood samples.

A widely-used liquid biopsy approach consists of genotyping the circulating tumour DNA (ctDNA) through sequencing of plasma/serum DNA.

Although useful in the context of metastatic disease (where the concentration of ctDNA is high), current liquid biopsy technologies show limited sensitivity of detection for the early detection of cancer, and low specificity, as cancer-related mutations accumulate in healthy tissues as part of the ageing process, thus making it difficult to distinguish these from tumour mutations, and because sequencing errors and true mutations show overlapping profiles.

Therefore, developing liquid biopsy protocols with increased sensitivity and specificity represents an urgent clinical need.

Aims I propose to harness extrachromosomal circular DNA (eccDNA) elements, which are circular DNA structures physically separated from the chromosomes of up to several Mbp long pervasive in human cancers, for liquid biopsy analysis. Methods Specifically, the protocol will consist of the following steps. 1.

Isolation and enrichment of eccDNA following standard protocols for plasmid purification using column chromatography. 2.

Amplification of eccDNA using rolling circle amplification, which consists of amplifying single or double-stranded eccDNA using the DNA polymerase from the bacteriophage F29. 3.

Whole-genome sequencing of (i) the primary tumour, (ii) a matched normal sample to enable the detection of tumour-specific mutations, (iii) the isolated and amplified eccDNA, and (iv) a plasma DNA sample containing both linear and circular DNA, which corresponds to standard liquid biopsy analysis based on unbiased sequencing of ctDNA. 4.

Sequencing data analysis to identify tumour-specific mutations mapping to eccDNA elements by focusing primarily on regions amplified to high copy number and showing discordant read clusters, as these are consistent with extrachromosomal DNA amplification.

The proposed approach will increase: (i) sensitivity, as only relevant DNA fragments are sequenced, and (ii) specificity, due to the fact that eccDNA elements contain tens of structural variants, which are infrequent sequencing errors, and contain cancer type-specific rearrangements (e.g., EGFR variant III mutant).

How the results of this research will be used.

In this pilot study we will focus on the analysis of sarcomas and glioblastomas, because there is strong evidence for the presence of eccDNA in these tumour types, and because developing liquid biopsy approaches for brain tumours to reduce the invasiveness of brain tumour biopsies remains an unmet clinical need.