Targeted KRAS mutation assessment on patient tumor histologic material in real time diagnostics.
Τίτλος | Targeted KRAS mutation assessment on patient tumor histologic material in real time diagnostics. |
Publication Type | Journal Article |
Year of Publication | 2009 |
Authors | Kotoula, V., Charalambous E., Biesmans B., Malousi A., Vrettou E., Fountzilas G., & Karkavelas G. |
Journal | PLoS One |
Volume | 4 |
Issue | 11 |
Pagination | e7746 |
Date Published | 2009 |
ISSN | 1932-6203 |
Λέξεις κλειδιά | Decision Support Techniques, DNA, Neoplasm, Exons, Genes, ras, Genotype, Humans, Mutation, Neoplasm Metastasis, Neoplasms, Prospective Studies, Quality Control, ras Proteins, Reverse Transcriptase Polymerase Chain Reaction, Sequence Analysis, DNA, Software |
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.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 detect99%) 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. |
DOI | 10.1371/journal.pone.0007746 |
Alternate Journal | PLoS ONE |
PubMed ID | 19888477 |
PubMed Central ID | PMC2768905 |