Detection of Circulating Tumor DNA in Patients with Pancreatic Cancer Using Digital Next-Generation Sequencing

Anne Macgregor-Das, Jun Yu, Koji Tamura, Toshiya Abe, Masaya Suenaga, Koji Shindo, Michael Borges, Chiho Koi, Shiro Kohi, Yoshihiko Sadakari, Marco Dal Molin, Jose A. Almario, Madeline Ford, Miguel Chuidian, Richard Burkhart, Jin He, Ralph H. Hruban, James R. Eshleman, Alison P. Klein, Christopher L. WolfgangMarcia I. Canto, Michael Goggins

Research output: Contribution to journalArticlepeer-review

3 Citations (Scopus)

Abstract

Circulating tumor DNA (ctDNA) measurements can be used to estimate tumor burden, but avoiding false-positive results is challenging. Herein, digital next-generation sequencing (NGS) is evaluated as a ctDNA detection method. Plasma KRAS and GNAS hotspot mutation levels were measured in 140 subjects, including 67 with pancreatic ductal adenocarcinoma and 73 healthy and disease controls. To limit chemical modifications of DNA that yield false-positive mutation calls, plasma DNA was enzymatically pretreated, after which DNA was aliquoted for digital detection of mutations (up to 384 aliquots/sample) by PCR and NGS. A digital NGS score of two SDs above the mean in controls was considered positive. Thirty-seven percent of patients with pancreatic cancer, including 31% of patients with stages I/II disease, had positive KRAS codon 12 ctDNA scores; only one patient had a positive GNAS mutation score. Two disease control patients had positive ctDNA scores. Low-normal–range digital NGS scores at mutation hotspots were found at similar levels in healthy and disease controls, usually at sites of cytosine deamination, and were likely the result of chemical modification of plasma DNA and NGS error rather than true mutations. Digital NGS detects mutated ctDNA in patients with pancreatic cancer with similar yield to other methods. Detection of low-level, true-positive ctDNA is limited by frequent low-level detection of false-positive mutation calls in plasma DNA from controls.

Original languageEnglish
Pages (from-to)748-756
Number of pages9
JournalJournal of Molecular Diagnostics
Volume22
Issue number6
DOIs
Publication statusPublished - Jun 2020
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Pathology and Forensic Medicine
  • Molecular Medicine

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