Targeted KRAS Mutation Assessment on Patient Tumor Histologic Material in Real Time Diagnostics

被引:74
作者
Kotoula, Vassiliki
Charalambous, Elpida
Biesmans, Bart
Malousi, Andigoni
Vrettou, Eleni
Fountzilas, George
Karkavelas, George
机构
[1] Department of Pathology, Papageorgiou Hospital, Aristotle University of Thessaloniki, Thessaloniki
[2] Department of Medical Informatics, Papageorgiou Hospital, Aristotle University of Thessaloniki, Thessaloniki
[3] Department of Medical Oncology, Papageorgiou Hospital, Aristotle University of Thessaloniki, Thessaloniki
[4] Department of Human Genetics, University of Leuven, Leuven
来源
PLOS ONE | 2009年 / 4卷 / 11期
关键词
METASTATIC COLORECTAL-CANCER; PARAFFIN-EMBEDDED TISSUES; K-RAS MUTATIONS; BREAST-CANCER; DNA PROBES; EGFR; CETUXIMAB; PCR; ASSAYS; CARCINOMA;
D O I
10.1371/journal.pone.0007746
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
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 QPCR 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/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.
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页数:13
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