Targeted next-generation sequencing: cell-free DNA profiles mirror the heterogeneity of single CTCs

David Guttery1,Karen Page1,Allison Hills2,Laura Woodley2,Genny Buson3,Raimo Tanzi3,Henrik Tommerup3,Justin Stebbing2,Charles Coombes2,Jacqueline Shaw1

1Department of Cancer Studies and Cancer Research UK Leicester Centre, Leicester, UK,2Imperial College, Department of Surgery and Cancer, Charing Cross Hospital, London, UK,3Silicon Biosystems, Bologna, Italy

Presenting date: Tuesday 3 November
Presenting time: 16.50-17.05


Cell-free DNA (cfDNA) and circulating tumour cells (CTCs) can provide a "liquid biopsy" as a surrogate for the tumour for real-time monitoring of patients with cancer. We aimed to compare single CTCs and a pool of CTCs isolated from CellSearch cassettes with matched cfDNA by targeted NGS.


CTCs were enriched and enumerated by CellSearch® from 7.5 ml of blood from 2 MBC patients with >100 CTCs. For one patient, five single CTCs and 5 lymphoctyes were isolated by DEParrayTM, and DNA was isolated and whole genome amplified (WGA) using the Ampli1 WGA kit (Silicon Biosystems). DNA was extracted from a pool of CTCs for the other patient. Matched cfDNA was analysed for both patients. Targeted NGS was performed with 2 amplicon panels (Ampli1 and a focused, custom mutation panel (1)) using the Ion PGMTM platform.


Molecular heterogeneity was observed in the 5 single CTCs. Of note, 2 CTCs had both a PIK3CA p.H1047R and an ESR1 p.E380Q mutation, which were absent from the other 3 CTCs, although 2 of these had a unique TP53 p.P72R mutation. A number of novel variants of unknown significance were also identified that were heterogeneous between CTCs. The matched cfDNA sample had all mutations found across the 5 CTCs, with no additional mutations unique to cfDNA. The other patient had the same ESR1 p.D538G mutation in DNA isolated from pooled CTCs and matched cfDNA, but also had a novel variant in exon 7 of FGFR1 in cfDNA.


These data confirm molecular heterogeneity of single CTCs and suggest cfDNA as a suitable biomarker for the genetic landscape of CTCs in MBC. Analysis of other patients and samples is currently ongoing.