title: Targeted KRAS mutation assessment on patient tumor histologic material in real time diagnostics.
abstract: BACKGROUND
  Testing for tumor specific mutations on routine formalin-fixed paraffin-embedded (FFPE) tissues may predict response to treatment in Medical Oncology and has already entered diagnostics, with KRAS mutation assessment as a paradigm. The highly sensitive real time PCR (Q-PCR) methods developed for this purpose are usually standardized under optimal template conditions. In routine diagnostics, however, suboptimal templates pose the challenge. Herein, we addressed the applicability of sequencing and two Q-PCR methods on prospectively assessed diagnostic cases for KRAS mutations.
  
  
  RESULTS
  Tumor FFPE-DNA from 135 diagnostic and 75 low-quality control samples was obtained upon macrodissection, tested for fragmentation and assessed for KRAS mutations with dideoxy-sequencing and with two Q-PCR methods (Taqman-minor-groove-binder [TMGB] probes and DxS-KRAS-IVD). Samples with relatively well preserved DNA could be accurately analyzed with sequencing, while Q-PCR methods yielded informative results even in cases with very fragmented DNA (p<0.0001) with 100% sensitivity and specificity vs each other. However, Q-PCR efficiency (Ct values) also depended on DNA-fragmentation (p<0.0001). Q-PCR methods were sensitive to detect<or=1% mutant cells, provided that samples yielded cycle thresholds (Ct)<29, but this condition was met in only 38.5% of diagnostic samples. In comparison, FFPE samples (>99%) could accurately be analyzed at a sensitivity level of 10% (external validation of TMGB results). DNA quality and tumor cell content were the main reasons for discrepant sequencing/Q-PCR results (1.5%).
  
  
  CONCLUSIONS
  Diagnostic targeted mutation assessment on FFPE-DNA is very efficient with Q-PCR methods in comparison to dideoxy-sequencing. However, DNA fragmentation/amplification capacity and tumor DNA content must be considered for the interpretation of Q-PCR results in order to provide accurate information for clinical decision making.
authors.affiliation: Department of Pathology, Papageorgiou Hospital, Aristotle University of Thessaloniki, School of Medicine, Thessaloniki, Greece. vkotoula@auth.gr
authors.forename: Vassiliki
authors.initials: V
authors.lastname: Kotoula
authors.forename: Elpida
authors.initials: E
authors.lastname: Charalambous
authors.forename: Bart
authors.initials: B
authors.lastname: Biesmans
authors.forename: Andigoni
authors.initials: A
authors.lastname: Malousi
authors.forename: Eleni
authors.initials: E
authors.lastname: Vrettou
authors.forename: George
authors.initials: G
authors.lastname: Fountzilas
authors.forename: George
authors.initials: G
authors.lastname: Karkavelas
mesh_terms: D004273:DNA, Neoplasm / Q000378:metabolism; D003661:Decision Support Techniques; D005091:Exons; D011905:Genes, ras; D005838:Genotype; D006801:Humans; D009154:Mutation; D009362:Neoplasm Metastasis; D009369:Neoplasms / Q000235:genetics* / Q000378:metabolism; D011446:Prospective Studies; D011786:Quality Control; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA; D012984:Software; D018631:ras Proteins / Q000235:genetics*
chemical_list: D004273:DNA, Neoplasm; D018631:ras Proteins