Individualized Mutation Detection in Circulating Tumor DNA for Monitoring Colorectal Tumor Burden Using a Cancer-Associated Gene Sequencing Panel

PLoS One. 2016 Jan 4;11(1):e0146275. doi: 10.1371/journal.pone.0146275. eCollection 2016.

Abstract

Background: Circulating tumor DNA (ctDNA) carries information on tumor burden. However, the mutation spectrum is different among tumors. This study was designed to examine the utility of ctDNA for monitoring tumor burden based on an individual mutation profile.

Methodology: DNA was extracted from a total of 176 samples, including pre- and post-operational plasma, primary tumors, and peripheral blood mononuclear cells (PBMC), from 44 individuals with colorectal tumor who underwent curative resection of colorectal tumors, as well as nine healthy individuals. Using a panel of 50 cancer-associated genes, tumor-unique mutations were identified by comparing the single nucleotide variants (SNVs) from tumors and PBMCs with an Ion PGM sequencer. A group of the tumor-unique mutations from individual tumors were designated as individual marker mutations (MMs) to trace tumor burden by ctDNA using droplet digital PCR (ddPCR). From these experiments, three major objectives were assessed: (a) Tumor-unique mutations; (b) mutation spectrum of a tumor; and (c) changes in allele frequency of the MMs in ctDNA after curative resection of the tumor.

Results: A total of 128 gene point mutations were identified in 27 colorectal tumors. Twenty-six genes were mutated in at least 1 sample, while 14 genes were found to be mutated in only 1 sample, respectively. An average of 2.7 genes were mutated per tumor. Subsequently, 24 MMs were selected from SNVs for tumor burden monitoring. Among the MMs found by ddPCR with > 0.1% variant allele frequency in plasma DNA, 100% (8 out of 8) exhibited a decrease in post-operation ctDNA, whereas none of the 16 MMs found by ddPCR with < 0.1% variant allele frequency in plasma DNA showed a decrease.

Conclusions: This panel of 50 cancer-associated genes appeared to be sufficient to identify individual, tumor-unique, mutated ctDNA markers in cancer patients. The MMs showed the clinical utility in monitoring curatively-treated colorectal tumor burden if the allele frequency of MMs in plasma DNA is above 0.1%.

Publication types

  • Clinical Trial
  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenocarcinoma / blood*
  • Adenocarcinoma / genetics
  • Adenocarcinoma / pathology
  • Adenocarcinoma / surgery
  • Adult
  • Aged
  • Alleles
  • Cell Line, Tumor
  • Colorectal Neoplasms / blood*
  • Colorectal Neoplasms / genetics
  • Colorectal Neoplasms / pathology
  • Colorectal Neoplasms / surgery
  • DNA Mutational Analysis*
  • DNA Primers
  • DNA, Neoplasm / blood*
  • Female
  • Genes, Neoplasm*
  • Humans
  • Leukocytes, Mononuclear / chemistry
  • Male
  • Middle Aged
  • Multiplex Polymerase Chain Reaction
  • Point Mutation*
  • Polymorphism, Single Nucleotide*
  • Sequence Analysis, DNA / instrumentation
  • Sequence Analysis, DNA / methods*
  • Tumor Burden*

Substances

  • DNA Primers
  • DNA, Neoplasm

Grants and funding

This work was supported by Keiryoukai Research Foundation No.125 of Iwate Medical University [KAS]; and Ministry of Education, Culture, Sports, Science and Technology, Japan for MIAST (Medical Innovation by Advanced Science and Technology) project of the grant-in-Aid for Strategic Research Foundation at Private Universities [S1001001 to SSN]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.