Targeted KRAS mutation assessment on patient tumor histologic material in real time diagnostics

PLoS One. 2009 Nov 4;4(11):e7746. doi: 10.1371/journal.pone.0007746.


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.

Methodology/principal findings: 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/significance: 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA, Neoplasm / metabolism
  • Decision Support Techniques
  • Exons
  • Genes, ras*
  • Genotype
  • Humans
  • Mutation*
  • Neoplasm Metastasis
  • Neoplasms / genetics*
  • Neoplasms / metabolism
  • Prospective Studies
  • Quality Control
  • Reverse Transcriptase Polymerase Chain Reaction / methods*
  • Sequence Analysis, DNA
  • Software
  • ras Proteins / genetics*


  • DNA, Neoplasm
  • ras Proteins