Multimodal cell-free DNA whole-genome analysis combined with TET-Assisted Pyridine Borane Sequencing is sensitive and reveals specific cancer signals

The analysis of circulating tumour DNA (ctDNA) promises to extend current tissue-specific cancer screening programmes to multi-cancer early detection and measurable disease monitoring to solid tumours using minimally invasive blood draws (liquid biopsies). Most studies so far have focussed on using targeted deep sequencing to detect the low-abundance, fragmented ctDNA. A few studies have integrated information from multiple modalities using shallow 1× WGS. Here, we developed an integrated bioinformatics pipeline for ctDNA detection based on whole genome TET-Assisted Pyridine Borane Sequencing (TAPS) of plasma samples sequenced at 80× or higher. We conducted a diagnostic accuracy study in a case-control cohort of patients presenting to the UK National Health Service’s (NHS) primary care pathway with non-specific symptoms of cancer, who either did not have cancer or who were subsequently diagnosed with cancer and referred to surgery with curative intent. TAPS is a base-level-resolution sequencing methodology for the detection of 5-methylcytosines and 5-hydro-methylcytosines. Unlike bisulfite-sequencing, the current established method for mapping epigenetic DNA modifications, TAPS is a non-destructive methodology, which only converts methylated cytosines and preserves DNA fragments over 10 kilobases long, thus opening the possibility of simultaneous methylome and genome analysis on the same sequencing data. The proposed methodology combines copy number aberrations and single nucleotide variants with methylation calls from TAPS-treated plasma from patients with Stage 1-4 colorectal (n=36), oesophageal (n=8), pancreatic (n=6), renal (n=5), ovarian (n=4) and breast (n=2) cancers. Plasma samples from 21 confirmed non-cancer controls were used for data denoising, while plasma samples from 9 additional agematched healthy controls were further used to establish the minimum level of detection. Copy number aberrations, single nucleotide variants, and methylation signals were independently analysed and combined in sample-specific scores, which quantify the levels of plasma ctDNA. Matched tumour samples were used for validation, not for guiding the analysis, imitating an early detection scenario. The detection threshold was set such that specificity was 100%, resulting in sensitivity of 85.2%. In silico experiments on high-fidelity synthetic data suggest excellent discriminatory capacity (AUC > 80%) at ctDNA fractions as low as 0.7%. Furthermore, we demonstrate successful tracking of tumour burden post-treatment and ctDNA shedding in precancerous adenomas in patients with colorectal cancer in the absence of a matched tumour biopsy. In summary, we developed and validated a pipeline for interrogating liquid biopsies using TAPS 80× or higher WGS that is ready for in-depth clinical evaluation both in multi-cancer screening of high-risk individuals and multi-cancer measurable disease monitoring. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This research was funded by Innovate UK and the National Institute for Health Research (NIHR) Oxford Biomedical Research Centre. We further acknowledge the contribution to this study made by the Oxford Centre for Histopathology Research and the Oxford Radcliffe Biobank, which are supported by the University of Oxford, the Ox-ford CRUK Cancer Centre and the NIHR Oxford Biomedical Research Centre (Molecular Diagnostics Theme/Multimodal Pathology Subtheme), and the NIHR CRN Thames Valley network. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health and Social Care. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The National Health Service (NHS) Health Research Authority South Central-Oxford C Research Ethics Committee approved this study, and all research was performed in accordance with relevant regulations and guidelines and with the Declaration of Helsinki. Written informed consent was obtained for patients recruited into the 100,000 Genomics England (GEL) pilot study and the rapid diagnostic clinic research pathway in Oxford called Suspected CANcer (SCAN) according to Oxford Radcliffe Biobank (ORB) guidelines (Oxford C Research Ethics Committee Number: 19/SC/0173). For the healthy controls, blood was received from Cambridge Bioscience Human Blood Products Supply Service, where comprehensive informed written consent was provided in accordance with UK ethics and consent regulations. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors