Sensitive Detection of KRAS Mutations in Archived Formalin-Fixed Paraffin-Embedded Tissue Using Mutant-Enriched PCR and Reverse-Hybridization

Abstract

Recently, evidence has emerged indicating that assessment of KRAS mutations before anti-epidermal growth factor receptor therapy improves outcome in patients with metastatic colorectal cancer (CRC). We report here a novel reverse-hybridization (RH) assay to screen for KRAS mutations in formalin-fixed paraffin-embedded colorectal tissue samples. We combined mutant-enriched PCR based on peptide nucleic acid clamping and RH of amplification products to nitrocellulose test strips that contained a parallel array of oligonucleotide probes targeting 10 frequent mutations in codons 12 and 13 of the KRAS gene. DNA mixing experiments, which included eight different tumor cell lines with known KRAS mutations, were performed to examine the sensitivity of mutation detection. All KRAS mutations present in tumor cell lines were unambiguously identified by the RH assay with 1% of each cell line DNA diluted in normal DNA. RH was then used to screen for KRAS mutations in 74 colorectal tumor and 4 normal control samples. Twenty-six (35%) of the 74 tumor samples showed KRAS mutations. No mutation was found in the four samples of normal colorectal tissue. DNA sequencing without previous mutant enrichment, however, failed to detect four (15%) out of 26 KRAS-positive formalin-fixed paraffin-embedded samples (FFPE). This finding suggests that even after microdissection, mutant sequences in a given DNA isolate can be rare and more sensitive methods are needed for mutation analysis.

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

“…With this technique, mutations were detected in samples containing as little as 1% tumor cells in a wild-type background. These results are in agreement with previous reports using cell lines 11 in which the same sensitivity was found for mutation detection.…”

“…The KRAS StripAssay as recently described by Ausch et al 11 was performed according to the manufacturer's instructions (Vienna Labs, Vienna, Austria). Briefly summarized, a PCR enriched for mutant KRAS alleles is performed.…”

“…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.…”

KRAS Testing: A Tool for the Implementation of Personalized Medicine

“…26,30,32,[34][35][36]50,51 Array/strip assays appear to reach the highest sensitivity, with some assays detecting 0.1% mutant DNA. [45][46][47][48][49] Conversely, HRM, SSCP, and DGGE often show relatively high false-positive rates compared to other techniques and often require additional testing with different methodologies to confirm the mutation detected. 24,26,30,38,[41][42][43][44] These techniques hold value for large-volume screening protocols, such as research applications, but provide little value in most molecular diagnostic laboratory applications.…”

“…Several studies have demonstrated high sensitivities and specificities of up to 100%. [45][46][47][48] Interestingly, like sequencing, this technique can detect rare cases where a given sample has more than one type of KRAS mutation, although it only detects those mutations present on the analysis strip. Due to the high sensitivity of the assays, this technique has been successfully applied to KRAS mutation detection in samples that are likely to contain large amounts of nonmutant DNA, including blood and feces.…”