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Development and Clinical Validation of Discriminatory Multitarget Digital Droplet PCR Assays for the Detection of Hot Spot KRAS and NRAS Mutations in Cell-Free DNA.
Journal article

Development and Clinical Validation of Discriminatory Multitarget Digital Droplet PCR Assays for the Detection of Hot Spot KRAS and NRAS Mutations in Cell-Free DNA.

  • Hussung S Department of Medicine I (Hematology, Oncology and Stem Cell Transplantation), Freiburg University Medical Center, Freiburg, Germany; German Cancer Consortium (DKTK) partner site Freiburg, Heidelberg, Germany.
  • Follo M Department of Medicine I (Hematology, Oncology and Stem Cell Transplantation), Freiburg University Medical Center, Freiburg, Germany.
  • Klar RFU Department of Medicine I (Hematology, Oncology and Stem Cell Transplantation), Freiburg University Medical Center, Freiburg, Germany.
  • Michalczyk S Department of Medicine I (Hematology, Oncology and Stem Cell Transplantation), Freiburg University Medical Center, Freiburg, Germany.
  • Fritsch K Department of Medicine I (Hematology, Oncology and Stem Cell Transplantation), Freiburg University Medical Center, Freiburg, Germany.
  • Nollmann F Department of Medicine I (Hematology, Oncology and Stem Cell Transplantation), Freiburg University Medical Center, Freiburg, Germany.
  • Hipp J Department of Surgery, Freiburg University Medical Center, Freiburg, Germany.
  • Duyster J Department of Medicine I (Hematology, Oncology and Stem Cell Transplantation), Freiburg University Medical Center, Freiburg, Germany; German Cancer Consortium (DKTK) partner site Freiburg, Heidelberg, Germany; Comprehensive Cancer Center Freiburg, Medical Center-University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany.
  • Scherer F Department of Medicine I (Hematology, Oncology and Stem Cell Transplantation), Freiburg University Medical Center, Freiburg, Germany.
  • von Bubnoff N Department of Medicine I (Hematology, Oncology and Stem Cell Transplantation), Freiburg University Medical Center, Freiburg, Germany; German Cancer Consortium (DKTK) partner site Freiburg, Heidelberg, Germany.
  • Boerries M German Cancer Consortium (DKTK) partner site Freiburg, Heidelberg, Germany; Comprehensive Cancer Center Freiburg, Medical Center-University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany; Institute of Medical Bioinformatics and Systems Medicine, University Medical Center Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany; German Cancer Research Center (DFKZ), Heidelberg, Germany.
  • Wittel U Department of Surgery, Freiburg University Medical Center, Freiburg, Germany.
  • Fritsch RM Department of Medicine I (Hematology, Oncology and Stem Cell Transplantation), Freiburg University Medical Center, Freiburg, Germany; German Cancer Consortium (DKTK) partner site Freiburg, Heidelberg, Germany; Department of Medical Oncology and Hematology, Zurich University Hospital, Zurich, Switzerland. Electronic address: ralph.fritsch@usz.ch.
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  • 2020-05-08
Published in:
  • The Journal of molecular diagnostics : JMD. - 2020
Pathology and Forensic Medicine
Molecular Medicine
English Detection and quantification of tumor-derived KRAS and NRAS mutations in plasma cell-free DNA (cfDNA) holds great potential for cancer diagnostics and treatment response monitoring. Because of high sensitivity, specificity, robustness, and affordability, digital droplet PCR (ddPCR) is ideally suited for this application but requires discriminatory multiplexing when used as screening assay. We therefore designed, optimized, and clinically validated mutation-specific locked nucleic acid-based ddPCR assays for 14 commonly occurring KRAS and NRAS mutations and assembled these assays into seven discriminatory multitarget screening assays covering two to six single-nucleotide variants each. Limit of detection, limit of blank, and interassay accuracy were determined. Assay performance and suitability for screening in cfDNA were validated with plasma samples from a clinically fully characterized cohort of pancreatic cancer patients and healthy controls. Limits of detection for single-target assays were between 0.0015% and 0.069% variant allele fraction, and between 0.022% and 0.16% for multitarget assays. Dilution linearity and interassay accuracy were excellent throughout (r2 > 0.99). Multitarget assay screening of cfDNA extracted from pancreatic cancer patients with unknown KRAS mutational status correctly identified single-nucleotide variants in 45 of 45 (100%) of tumor-derived cell-free DNA-positive samples. In summary, we herein present and clinically validate generic single-target and discriminatory multitarget ddPCR assays for KRAS and NRAS hot spot mutations with broad applicability for clinical and translational research.
Language
  • English
Open access status
closed
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Persistent URL
https://folia.unifr.ch/global/documents/226209
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