title: Individualized Mutation Detection in Circulating Tumor DNA for Monitoring Colorectal Tumor Burden Using a Cancer-Associated Gene Sequencing Panel.
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.
  
  
  METHODS
  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%.
authors.affiliation: Molecular Therapeutics Laboratory, Department of Surgery, Iwate Medical University School of Medicine, Morioka, Japan.|Department of Surgery, Iwate medical University School of Medicine, Morioka, Japan.
authors.forename: Kei A
authors.initials: KA
authors.lastname: Sato
authors.affiliation: Division of Biomedical Information Analysis, Iwate Tohoku Medical Megabank Organization, Iwate Medical University, Yahaba, Japan.
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Hachiya
authors.affiliation: Department of Surgery, Iwate medical University School of Medicine, Morioka, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Iwaya
authors.affiliation: Molecular Therapeutics Laboratory, Department of Surgery, Iwate Medical University School of Medicine, Morioka, Japan.|Department of Surgery, Iwate medical University School of Medicine, Morioka, Japan.|MIAST (Medical Innovation by Advanced Science and Technology) Project, Iwate Medical University School, Morioka, Japan.|Institute of Biomedical Science, Iwate Medical University, Yahaba, Japan.
authors.forename: Kohei
authors.initials: K
authors.lastname: Kume
authors.affiliation: Department of Surgery, Iwate medical University School of Medicine, Morioka, Japan.
authors.forename: Teppei
authors.initials: T
authors.lastname: Matsuo
authors.affiliation: Division of Gastroenterology and Hepatology, Department of Internal Medicine, Iwate Medical University School of Medicine, Morioka, Japan.
authors.forename: Keisuke
authors.initials: K
authors.lastname: Kawasaki
authors.affiliation: Division of Gastroenterology and Hepatology, Department of Internal Medicine, Iwate Medical University School of Medicine, Morioka, Japan.
authors.forename: Yukito
authors.initials: Y
authors.lastname: Abiko
authors.affiliation: Division of Gastroenterology and Hepatology, Department of Internal Medicine, Iwate Medical University School of Medicine, Morioka, Japan.
authors.forename: Risaburo
authors.initials: R
authors.lastname: Akasaka
authors.affiliation: Division of Gastroenterology and Hepatology, Department of Internal Medicine, Iwate Medical University School of Medicine, Morioka, Japan.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Matsumoto
authors.affiliation: Department of Surgery, Iwate medical University School of Medicine, Morioka, Japan.
authors.forename: Koki
authors.initials: K
authors.lastname: Otsuka
authors.affiliation: Molecular Therapeutics Laboratory, Department of Surgery, Iwate Medical University School of Medicine, Morioka, Japan.|Department of Surgery, Iwate medical University School of Medicine, Morioka, Japan.|MIAST (Medical Innovation by Advanced Science and Technology) Project, Iwate Medical University School, Morioka, Japan.|Institute of Biomedical Science, Iwate Medical University, Yahaba, Japan.|Department of Surgery, Iwate Medical University School of Dentistry, Morioka, Japan.
authors.forename: Satoshi S
authors.initials: SS
authors.lastname: Nishizuka
mesh_terms: D000230:Adenocarcinoma / Q000097:blood* / Q000235:genetics / Q000473:pathology / Q000601:surgery; D000328:Adult; D000368:Aged; D000483:Alleles; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000097:blood* / Q000235:genetics / Q000473:pathology / Q000601:surgery; D004252:DNA Mutational Analysis; D017931:DNA Primers; D004273:DNA, Neoplasm / Q000097:blood*; D005260:Female; D052138:Genes, Neoplasm; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000737:chemistry; D008297:Male; D008875:Middle Aged; D060885:Multiplex Polymerase Chain Reaction; D017354:Point Mutation; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA / Q000295:instrumentation / Q000379:methods*; D047368:Tumor Burden
chemical_list: D017931:DNA Primers; D004273:DNA, Neoplasm