Abstract 3960: Combined circulating tumour cell (CTC) and circulating tumor DNA (ctDNA) analysis of blood from patients with pancreatic cancer
Authors: Brady Ged , Rothwell Dominic , Ayub Mahmood , Smith Nigel , Mohan Sumitra , Chudziak Jakub , Aung Kyaw , Hubner Richard , Miller Crispin , Backen Alison , Leong Hui Sun , Gulati Sakshi , Kim Chang Sik , Lamarca Angela , McNamara Mairéad , Valle Juan , Dive Caroline 
Published: July 2016

Abstract
Background
The challenge to improve outcomes for patients diagnosed with advanced pancreatic cancer remains very real with only small improvements in median survival gained by the use of systemic chemotherapy and little improvement in 5-year survival over the past decades. The advent of next generation sequencing (NGS) of tumor nucleic acids has opened up the possibility of improving outcomes through personalized therapies selected on the basis of tumor genetics.
Whilst NGS biomarkers can be measured in tumor biopsies sampled shortly before and after treatment this is often not practical for ethical, logistical and simple lack of availability. To circumvent problems associated with tumor sampling we have developed and evaluated blood-borne nucleic acids biomarkers for patients diagnosed with advanced pancreatic cancer.
Approach
We developed a NGS circulating free DNA (cfDNA) analysis pipeline based on the generation of whole genome NGS libraries followed by sequencing of over 600 cancer-associated genes using Agilent SureSelect. Using whole blood collected with Streck cell free DNA blood collection tubes (cfDNA BCT) we optimised combined circulating tumor cell (CTC) enrichment and cfDNA isolation. Quantitative measurements of KRAS mutations using both NGS and droplet digital PCR (ddPCR) were used to compare the tumor component present in both CTCs and cfDNA. We evaluated the overall approach using External Quality Assessment (EQA) controls and over 50 advanced pancreatic cancer patient samples. For CTC analysis we also compared epitope dependent (CellSearch) and independent (Parsortix) enrichment.
Results
After applying our NGS pipeline to 5 EQA genomic controls we identified all 14 known mutations correctly indicating high sensitivity and specificity. Both ddPCR and NGS identified KRAS mutations in patient cfDNA with a higher success rate seen for ddPCR consistent with the higher sensitivity of this methodology. From the NGS output additional mutations were detected in samples which either harboured or lacked detectable KRAS mutations. Consistent with previous observations CellSearch CTCs were detected at low levels in around 20% of patients with a similar frequency seen in initial analysis of CTCs obtained by epitope independent enrichment (Parsortix). In general KRAS mutations were detected at a higher level in patient cfDNA compared to enriched CTCs although some patients showed detectable CTC KRAS mutations with no KRAS mutations detected in their cfDNA by either ddPCR or NGS. Ongoing analysis is aimed at establishing if the molecular observations correlate with clinical outcome.
Conclusion
Combined CTC enrichment and cfDNA isolation is readily achievable using a single Streck cfDNA BCT. Results indicate that combined CTC and cfDNA analysis is more sensitive than either approach alone.
Citation Format: Ged Brady, Dominic Rothwell, Mahmood Ayub, Nigel Smith, Sumitra Mohan, Jakub Chudziak, Kyaw Aung, Richard Hubner, Crispin Miller, Alison Backen, Hui Sun Leong, Sakshi Gulati, Chang Sik Kim, Angela Lamarca, Mairéad McNamara, Juan Valle, Caroline Dive. Combined circulating tumour cell (CTC) and circulating tumor DNA (ctDNA) analysis of blood from patients with pancreatic cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3960.