Abstract
BackgroundTesting 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 FindingsTumor 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 ≤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/SignificanceDiagnostic 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.
Highlights
Based on accumulated knowledge about tumor biology, newer drugs are meant to treat cancer in a more rational way than classic chemotherapy, i.e., by targeting specific molecules and pathways that are essential for promoting tumor growth, maintenance and metastasis
As measured with UV-spectrometry, values corresponding to DNA quantity did not differ between groups A and B (Table 1); the samples of the two groups, differed substantially in the obtained absorbance values (A260/280 ratios), which correspond to DNA purity (Mann-Whitney p,0.0001)
This study shows that the assessment of DNA fragmentation provides important information on the amplification capacity of the extracted formalin-fixed paraffin-embedded (FFPE)-DNA and on the reliability of the obtained results, in line with previous reports on methods involving relatively long PCR products [30,31,45] and short ones, as is the case with Q-PCR assays [45]
Summary
Based on accumulated knowledge about tumor biology, newer drugs are meant to treat cancer in a more rational way than classic chemotherapy, i.e., by targeting specific molecules and pathways that are essential for promoting tumor growth, maintenance and metastasis. In this context, EGFR, a HER family receptor tyrosine kinase, has emerged as a major molecular target. We addressed the applicability of sequencing and two Q-PCR methods on prospectively assessed diagnostic cases for KRAS mutations
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
