Detection of KRAS mutations in liquid biopsies from metastatic colorectal cancer patients using droplet digital PCR, Idylla, and next generation sequencing

PLoS One. 2020 Nov 25;15(11):e0239819. doi: 10.1371/journal.pone.0239819. eCollection 2020.


Circulating tumor DNA (ctDNA) is released from cancer cells and oncogenic mutations in ctDNA can be measured from plasma samples. Droplet digital PCR (ddPCR) is a sensitive and specific method for the detection of mutations in ctDNA. We analyzed serial plasma samples (n = 80) from ten metastatic colorectal cancer (mCRC) patients with a known KRAS mutation in their primary tumor. The patients were undergoing oncological treatment with bevacizumab in combination with alternating capecitabine and oxaliplatin or irinotecan. Baseline ddPCR KRAS mutation allele frequency (MAF) values ranged from 0% to 63%. The first radiologic response evaluation criteria in solid tumors (RECIST) evaluation was performed 45-63 days after the initiation of treatment, and by this time three patients had an undetectable level of KRAS mutation, one had a MAF value of 0.5%, and one had a MAF value of 3% that had been reduced by 95% from the baseline value. In three of these patients the RECIST assessment was stable disease and in two partial response. In seven patients, ddPCR MAF values increased before radiological disease progression or death, while one patient remained disease-free with an undetectable KRAS mutation level. Next, we analyzed all available plasma samples with the Idylla ctKRAS system (n = 60), and found that the overall degree of agreement between ddPCR and Idylla was almost perfect (kappa value = 0.860). We used next-generation sequencing (NGS) to detect treatment-induced mutations in the last serial plasma sample of each patient, but were unable to find any new mutations when compared to the primary tumor. This study shows that ddPCR and Idylla are equally efficient for the detection of KRAS mutations in the liquid biopsies from mCRC patients and that ctDNA may indicate the disappearance of treatment responsive KRAS positive mCRC clones and serve as an early sign of disease progression.

Publication types

  • Clinical Trial, Phase II
  • Multicenter Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Aged
  • Antineoplastic Combined Chemotherapy Protocols / therapeutic use
  • Bevacizumab / therapeutic use
  • Biomarkers, Tumor / genetics*
  • Capecitabine / therapeutic use
  • Circulating Tumor DNA / blood
  • Circulating Tumor DNA / genetics*
  • Colonic Neoplasms / drug therapy
  • Colonic Neoplasms / genetics*
  • DNA Mutational Analysis / methods*
  • Female
  • Gene Frequency
  • High-Throughput Nucleotide Sequencing / methods
  • Humans
  • Irinotecan / therapeutic use
  • Liquid Biopsy / methods
  • Male
  • Middle Aged
  • Oxaliplatin / therapeutic use
  • Polymerase Chain Reaction / methods
  • Proto-Oncogene Proteins p21(ras) / genetics*


  • Biomarkers, Tumor
  • Circulating Tumor DNA
  • KRAS protein, human
  • Oxaliplatin
  • Bevacizumab
  • Capecitabine
  • Irinotecan
  • Proto-Oncogene Proteins p21(ras)

Grants and funding

AR received grants from the Finnish Cancer Foundation, Finska Läkaresällskapet, Helsinki University Central Hospital Research Funds, the Sigrid Jusélius Foundation, the University of Helsinki, and an unrestricted grant from Amgen (Research Institute HUCH (Helsinki University Central Hospital) #70045). AKA and KA also received grants from Helsinki University Central Hospital Research Funds. PO received grants from the Finnish Cancer Foundation, Finska Läkaresällskapet, the Relanders Foundation, the Competitive State Research Financing of the Expert Responsibility Area of Tampere, Tampere University Hospital, the Tampere University Hospital Foundation. The AXOAXI trial is an investigator initiated study and was supported by an unrestricted grant from Roche Finland (Research Institute HUCH (Helsinki University Central Hospital) #3507 / ML25345) (PO). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.