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

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.

SNaPshot and StripAssay as Valuable Alternatives to Direct Sequencing for KRAS Mutation Detection in Colon Cancer Routine Diagnostics

“…8 Finally, real-time allelic discrimination could also be a good alternative for direct sequencing because of the rapidity and high sensitivity of the technique; however, the difficulty of multiplexing and the similarity between the probes lead to higher DNA input and a high risk of decreased specificity due to cross reactivity of the different probes. 10 When considering all aspects, we conclude that for colon cancer diagnostics, in which sensitivity is generally not an issue, and when capillary electrophoresis facilities are already available, SNaPshot can be as valuable as direct sequencing. Workflow, time to results, hands-on time, and costs do not vary much between both techniques.…”

“…To reliably test a sample, at least 20% to 30% of tumor cells are needed. To date, there are several alternative assays available for (KRAS) mutation detection, including "homebrew" assays, such as high-resolution melting curve analysis (HRM), 7 pyrosequencing, 8 single nucleotide primer extension assay, 9 and allele-specific real-time PCR, 10 and commercially available assays, such as reverse hybridization test KRAS StripAssay (Vienna Labs, Vienna, Austria) 11 and real-time PCR-based TheraScreen (Roche Diagnostics, Almere, the Netherlands); all these assays greatly differ in sensitivity, specificity, DNA input, time to results, hands-on time, flexibility, workload, and costs. The single nucleotide primer extension (SNaPshot) assay is a home-brew, flexible assay, which might be easily extendable to other biomarkers, whereas from the commercially available assays, the KRAS StripAssay claims to be fast and very sensitive.…”

Increased sensitivity of KRAS mutation detection by high-resolution melting analysis of COLD-PCR products

“…The sensitivity of the TheraScreen ® kit is reported by the company to be 1% depending on the total amount of DNA used (http://www.dxsdiagnostics.com). However, it has been reported that only a minority of diagnostic samples derived from FFPE tissues can be analyzed at this level of sensitivity (Kotoula, et al, 2009). …”

Optimizing nucleic acid extraction from thyroid fine-needle aspiration cells in stained slides, formalin-fixed/paraffin-embedded tissues, and long-term stored blood samples

“…In molecular endocrinology, these approaches are of enormous importance for the diagnosis of inherited disease-causing mutations and/or somatic mutations. Therefore, the adequate isolation of nucleic acids from peripheral blood, fine-needle aspiration (FNA) cells in stained slides, and fresh and formalin-fixed/paraffinembedded (FFPE) tissues is crucial to ensure the success of these techniques, posing a challenge for routine DNA extraction protocols, especially when blood samples and FFPE tissues have been stored for long period, and when no other samples can be collected from patients lost to follow-up (2,3).…”

“…Furthermore, obtaining DNA and RNA from FFPE samples and stained slides is always a challenge because fixation, embedding, staining, and extraction methods generally inhibit nucleic acid retrieval from the samples (3). On the other hand, although formalin fixation may degrade nucleic acids, it also deactivates nucleases and, thus, has a stabilizing effect (3).…”