Comparison of multiplex PCR and hybrid capture assays targeting patient-specific genomic alterations for sensitive detection of circulating tumour DNA in Stage IA-IV breast cancer - 11th International Symposium on Circulating Nucleic Acids in Plasma and Serum (CNAPS)

Angela Santonja Climent ^1,2 Wendy Cooper ^1,2 Katrin Heider ^1,2 Stephen-John Sammut ^1,2,3 James Morris ^1,2 Heather Biggs ^3,4 Abigail Edwards ^1,2 Dominique-Laurent Couturier ^1,2 Chris Boursnell ^1,2 Paul Edwards ^1,2 Carlos Caldas ^1,2,3,4 Nitzan Rosenfeld ^1,2 Jean Abraham ^3,4 Davina Gale ^1,2

¹Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, UK
²Cancer Research UK Cambridge Centre, University of Cambrdige, Cambridge, UK
³Department of Oncology, Cambridge University Hospitals NHS Foundation Trust, Addenbrooke’s Hospital, Cambridge, UK
⁴Cambridge Breast Unit, Cambridge University Hospitals NHS Foundation Trust, Addenbrooke’s Hospital, Cambridge, UK

Levels ctDNA are low in early stage cancer, but can potentially be detected using patient-specific sequencing assays. Structural variants (SVs) are present in most tumours, and can be assayed with lower background noise compared to single-nucleotide variants (SNVs). However, SNVs are more prevalent and can be analysed in larger numbers. To compare different approaches to detect ctDNA in plasma, we developed 1) multiplex PCR assays targeting SVs; 2) custom hybrid capture assays targeting both SVs and SNVs, and 3) shallow whole genome sequencing (sWGS, median coverage = 1.6x) to identify somatic copy number aberrations (SCNAs).

To identify genomic alterations, tumour and germline DNA were first analysed by whole genome sequencing (median coverage = 116x and 39x, respectively). Patient-specific multiplex PCR assays were developed targeting 21-49 SVs (median=31), and hybrid capture assays were designed targeting 31-156 SVs (median=41) and 1924-7543 SNVs (median=4654). The assays were used to monitor ctDNA in serial plasma samples from seven patients with Stage IA-IV breast cancer. To enhance sensitivity of detection of SNVs, signal was aggregated and enriched using an in-house INVAR analysis pipeline (INtegration of VAriant Reads).

When ctDNA was detected, similar levels were observed using both multiplex PCR and hybrid capture. sWGS was the least sensitive technology, identifying ctDNA in ~10% of the plasma samples. Using the capture-based approach, SNVs could be detected down to a few parts per million, benefitting from the large number of targeted mutations. For analysis of SVs, multiplex PCR proved to be more sensitive than custom capture in detecting ctDNA, even with less input DNA.

Using a low number of SVs, multiplex PCR provided higher sensitivity than the capture assay targeting the same loci. Overall, however, capture combined with INVAR analysis was the most sensitive method to detect ctDNA when thousands of patient-specific SNVs were assayed.