EACR Liquid biopsies

Genome-wide cell free DNA (cfDNA) methylation profiling, including the in-house developed assay, T7-MBD-seq, has demonstrated potential in the detection and molecular subtyping of cancer. Using this assay, a tissue-of-origin blood test has been developed to provide the prediction of the primary site for patients with Cancers of Unknown Primary (CUP) utilising the tissue specificity of DNA methylation patterns. Proof-of-concept data from both known cancer types and patients with CUP will be presented, demonstrating overall multiclass sensitivity of 84.6% and accuracy of 96.8%. Predictions were made for 78.0% of CUP cases, of which 71.9% were clinically consistent with a subsequent or suspected primary tumour diagnosis.

However, current approaches for cfDNA methylation analysis as a single -omic layer have limitations in terms of sensitivity for a growing range of clinical applications, and do not inform on the clinically actionable genomic alterations of the patients. The duet evoC technology, also known as 6-base chemistry sequencing, combines generation of DNA copies with enzymatic conversion to enable the measurement of both DNA methylation, hydroxymethylation and mutations from the same sample in the same assay. This approach is being used to develop circulating biomarkers for a number of cancer types including oesophago-gastric adenocarcinoma, a cancer type which has been reported to be a low shedder of cfDNA. Preliminary findings exploring the use of cfDNA methylation alone and in combination with added hydroxymethylation, mutations and fragmentomic features will be presented.

Authors: A. Tivey, A. Clipson, M. Halford, D. A. Onuselogu, P. Harker, M. Hossain, S. M. Hill, S. Makeev, D. Rothwell, C.Dive, R. Lee, F. Mouliere

Patient outcomes in advanced melanoma have been transformed by immune checkpoint inhibitors (ICIs). Patients who develop resistance have limited treatment options; there is a need for biomarkers to identify these patients and refine treatment decisions.

A multi-omic cell-free DNA (cfDNA) based ‘liquid biopsy’ could offer insight into tumour and immune changes at baseline and throughout therapy. Genomic analyses could inform on tumour mutational signal and neo-antigen load. Methylation analyses can be used to deconvolute the cfDNA cell-of-origin which may inform on immune cell turnover and normal tissue toxicity. Multi-omic profiling could enhance liquid biopsy but can involve multiplication of experiments and workflows reducing its potential to be clinically implemented.

We are evaluating the duet evoC technology which notably provides cfDNA genomic, methylation and hydroxymethylation information from a single workflow. We are comparing this 6-base genome sequencing with targeted sequencing and capture-based methylation enrichment (T7-MBD-seq). Initial analysis of cfDNA samples from patients with advanced cancer has shown concordance of tumour fraction and methylation profiling when assessed by the different methods.  At low coverage (~3x) WGS, 88% mutations with variant allele frequency of ≥20% by targeted sequencing were correctly identified by duet evoC. Both de novo identified differentially methylated regions and regions from an external reference atlas were able to distinguish samples from patients with melanoma from other samples using this technology.

Future objectives include comparing duet evoC with T7-MBD-seq for deconvoluting cell-of-origin from in-vitro and in-silico mixture sets of reference material, and comparing the methylation and hydroxymethylation profiles in longitudinal samples from patients with melanoma treated with ICIs.

In conclusion, we are assessing the potential of a multi-omic assay that can capture both genomic and epigenomic features of tumour- and immune-derived cfDNA. This will enable us to characterise comprehensively the cfDNA phenotypes associated with ICI response and resistance in patients with melanoma.