title: Multiplex KRASG12/G13 mutation testing of unamplified cell-free DNA from the plasma of patients with advanced cancers using droplet digital polymerase chain reaction.
abstract: Cell-free DNA (cfDNA) from plasma offers easily obtainable material for KRAS mutation analysis. Novel, multiplex, and accurate diagnostic systems using small amounts of DNA are needed to further the use of plasma cfDNA testing in personalized therapy.
  
  
  
  Samples of 16 ng of unamplified plasma cfDNA from 121 patients with diverse progressing advanced cancers were tested with a KRASG12/G13 multiplex assay to detect the seven most common mutations in the hotspot of exon 2 using droplet digital polymerase chain reaction (ddPCR). The results were retrospectively compared to mutation analysis of archival primary or metastatic tumor tissue obtained at different points of clinical care.
  
  
  
  Eighty-eight patients (73%) had KRASG12/G13 mutations in archival tumor specimens collected on average 18.5 months before plasma analysis, and 78 patients (64%) had KRASG12/G13 mutations in plasma cfDNA samples. The two methods had initial overall agreement in 103 (85%) patients (kappa, 0.66; ddPCR sensitivity, 84%; ddPCR specificity, 88%). Of the 18 discordant cases, 12 (67%) were resolved by increasing the amount of cfDNA, using mutation-specific probes, or re-testing the tumor tissue, yielding overall agreement in 115 patients (95%; kappa 0.87; ddPCR sensitivity, 96%; ddPCR specificity, 94%). The presence of ≥ 6.2% of KRASG12/G13 cfDNA in the wild-type background was associated with shorter survival (P = 0.001).
  
  
  
  Multiplex detection of KRASG12/G13 mutations in a small amount of unamplified plasma cfDNA using ddPCR has good sensitivity and specificity and good concordance with conventional clinical mutation testing of archival specimens. A higher percentage of mutant KRASG12/G13 in cfDNA corresponded with shorter survival.
authors.affiliation: Department of Investigational Cancer Therapeutics (Phase I Clinical Trials Program), The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
authors.forename: F
authors.initials: F
authors.lastname: Janku
authors.affiliation: Department of Investigational Cancer Therapeutics (Phase I Clinical Trials Program), The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
authors.forename: H J
authors.initials: HJ
authors.lastname: Huang
authors.affiliation: Department of Investigational Cancer Therapeutics (Phase I Clinical Trials Program), The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
authors.forename: T
authors.initials: T
authors.lastname: Fujii
authors.affiliation: Bio-Rad Laboratories, Pleasanton, CA, USA.|Holden Comprehensive Cancer Center, University of Iowa Health Care, Iowa City, IA, USA.
authors.forename: D N
authors.initials: DN
authors.lastname: Shelton
authors.affiliation: Department of Investigational Cancer Therapeutics (Phase I Clinical Trials Program), The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
authors.forename: K
authors.initials: K
authors.lastname: Madwani
authors.affiliation: Department of Investigational Cancer Therapeutics (Phase I Clinical Trials Program), The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
authors.forename: S
authors.initials: S
authors.lastname: Fu
authors.affiliation: Department of Investigational Cancer Therapeutics (Phase I Clinical Trials Program), The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
authors.forename: A M
authors.initials: AM
authors.lastname: Tsimberidou
authors.affiliation: Department of Investigational Cancer Therapeutics (Phase I Clinical Trials Program), The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
authors.forename: S A
authors.initials: SA
authors.lastname: Piha-Paul
authors.affiliation: Department of Investigational Cancer Therapeutics (Phase I Clinical Trials Program), The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
authors.forename: J J
authors.initials: JJ
authors.lastname: Wheler
authors.affiliation: Department of Investigational Cancer Therapeutics (Phase I Clinical Trials Program), The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
authors.forename: R G
authors.initials: RG
authors.lastname: Zinner
authors.affiliation: Department of Investigational Cancer Therapeutics (Phase I Clinical Trials Program), The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
authors.forename: A
authors.initials: A
authors.lastname: Naing
authors.affiliation: Department of Investigational Cancer Therapeutics (Phase I Clinical Trials Program), The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
authors.forename: D S
authors.initials: DS
authors.lastname: Hong
authors.affiliation: Department of Investigational Cancer Therapeutics (Phase I Clinical Trials Program), The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
authors.forename: D D
authors.initials: DD
authors.lastname: Karp
authors.affiliation: Department of Investigational Cancer Therapeutics (Phase I Clinical Trials Program), The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
authors.forename: G
authors.initials: G
authors.lastname: Cabrilo
authors.affiliation: Department of Gastrointestinal Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
authors.forename: E S
authors.initials: ES
authors.lastname: Kopetz
authors.affiliation: Department of Investigational Cancer Therapeutics (Phase I Clinical Trials Program), The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
authors.forename: V
authors.initials: V
authors.lastname: Subbiah
authors.affiliation: Hematopathology, Molecular Diagnostic Laboratory, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
authors.forename: R
authors.initials: R
authors.lastname: Luthra
authors.affiliation: Department of Gastrointestinal Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
authors.forename: B K
authors.initials: BK
authors.lastname: Kee
authors.affiliation: Department of Gastrointestinal Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
authors.forename: C
authors.initials: C
authors.lastname: Eng
authors.affiliation: Department of Gastrointestinal Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
authors.forename: V K
authors.initials: VK
authors.lastname: Morris
authors.affiliation: Bio-Rad Laboratories, Pleasanton, CA, USA.
authors.forename: G A
authors.initials: GA
authors.lastname: Karlin-Neumann
authors.affiliation: Department of Investigational Cancer Therapeutics (Phase I Clinical Trials Program), The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
authors.forename: F
authors.initials: F
authors.lastname: Meric-Bernstam
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D014408:Biomarkers, Tumor / Q000097:blood* / Q000235:genetics; D000073888:Cell-Free Nucleic Acids / Q000097:blood* / Q000235:genetics; D000074141:Circulating Tumor DNA / Q000097:blood / Q000235:genetics; D004252:DNA Mutational Analysis; D018572:Disease-Free Survival; D005091:Exons / Q000235:genetics; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D060885:Multiplex Polymerase Chain Reaction; D009369:Neoplasms / Q000097:blood* / Q000235:genetics; D016283:Proto-Oncogene Proteins p21(ras) / Q000097:blood / Q000235:genetics*
chemical_list: D014408:Biomarkers, Tumor; D000073888:Cell-Free Nucleic Acids; D000074141:Circulating Tumor DNA; C117307:KRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)
keywords:  cell-free DNA; KRAS; droplet digital PCR; multiplex