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@ARTICLE{Rassner:274559,
      author       = {M. Rassner and S. Waldeck$^*$ and M. Follo and S. Jilg and
                      U. Philipp and M. Jolic and J. Wehrle and P. J. Jost and C.
                      Peschel and A. L. Illert$^*$ and J. Duyster$^*$ and F.
                      Scherer$^*$ and N. von Bubnoff$^*$},
      title        = {{D}evelopment of {H}ighly {S}ensitive {D}igital {D}roplet
                      {PCR} for {D}etection of c{KIT} {M}utations in {C}irculating
                      {F}ree {DNA} {T}hat {M}ediate {R}esistance to {TKI}
                      {T}reatment for {G}astrointestinal {S}tromal {T}umor
                      ({GIST}).},
      journal      = {International journal of molecular sciences},
      volume       = {24},
      number       = {6},
      issn         = {1422-0067},
      address      = {Basel},
      publisher    = {Molecular Diversity Preservation International},
      reportid     = {DKFZ-2023-00636},
      pages        = {5411},
      year         = {2023},
      abstract     = {Mutations in cKIT or PDGFRA are found in up to $90\%$ of
                      patients with gastrointestinal stromal tumors (GISTs).
                      Previously, we described the design, validation, and
                      clinical performance of a digital droplet (dd)PCR assay
                      panel for the detection of imatinib-sensitive cKIT and
                      PDFGRA mutations in circulating tumor (ct)DNA. In this
                      study, we developed and validated a set of ddPCR assays for
                      the detection of cKIT mutations mediating resistance to cKIT
                      kinase inhibitors in ctDNA. In addition, we cross-validated
                      these assays using next generation sequencing (NGS).We
                      designed and validated five new ddPCR assays to cover the
                      most frequent cKIT mutations mediating imatinib resistance
                      in GISTs. For the most abundant
                      imatinib-resistance-mediating mutations in exon 17, a
                      drop-off, probe-based assay was designed. Dilution series
                      (of decreasing mutant (MUT) allele frequency spiked into
                      wildtype DNA) were conducted to determine the limit of
                      detection (LoD). Empty controls, single wildtype controls,
                      and samples from healthy individuals were tested to assess
                      specificity and limit of blank (LoB). For clinical
                      validation, we measured cKIT mutations in three patients and
                      validated results using NGS.Technical validation
                      demonstrated good analytical sensitivity, with a LoD ranging
                      between $0.006\%$ and $0.16\%$ and a LoB ranging from 2.5 to
                      6.7 MUT fragments/mL. When the ddPCR assays were applied to
                      three patients, the abundance of ctDNA in serial plasma
                      samples reflected the individual disease course, detected
                      disease activity, and indicated resistance mutations before
                      imaging indicated progression. Digital droplet PCR showed
                      good correlation to NGS for individual mutations, with a
                      higher sensitivity of detection.This set of ddPCR assays,
                      together with our previous set of cKIT and PDGFRA mutations
                      assays, allows for dynamic monitoring of cKIT and PDGFRA
                      mutations during treatment. Together with NGS, the GIST
                      ddPCR panel will complement imaging of GISTs for early
                      response evaluation and early detection of relapse, and thus
                      it might facilitate personalized decision-making.},
      keywords     = {GIST (Other) / PDGFRA (Other) / biomarkers (Other) / cKIT
                      (Other) / ctDNA (Other) / ddPCR (Other) / liquid biopsy
                      (Other)},
      cin          = {FR01},
      ddc          = {540},
      cid          = {I:(DE-He78)FR01-20160331},
      pnm          = {899 - ohne Topic (POF4-899)},
      pid          = {G:(DE-HGF)POF4-899},
      typ          = {PUB:(DE-HGF)16},
      pubmed       = {pmid:36982486},
      doi          = {10.3390/ijms24065411},
      url          = {https://inrepo02.dkfz.de/record/274559},
}