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

Abstract

Although direct sequencing is the gold standard for KRAS mutation detection in routine diagnostics, it remains laborious, time consuming, and not very sensitive. Our objective was to evaluate SNaPshot and the KRAS StripAssay as alternatives to sequencing for KRAS mutation detection in daily practice. KRAS exon 2-specific PCR followed by sequencing or by a SNaPshot reaction was performed. For the StripAssay, a mutant-enriched PCR was followed by hybridization to KRAS-specific probes bound to a nitrocellulose strip. To test sensitivities, dilution series of mutated DNA in wild-type DNA were made. Additionally, direct sequencing and SNaPshot were evaluated in 296 colon cancer samples. Detection limits of direct sequencing, SNaPshot, and StripAssay were 20%, 10%, and 1% tumor cells, respectively. Direct sequencing and SNaPshot can detect all 12 mutations in KRAS codons 12 and 13, whereas the StripAssay detects 10 of the most frequent ones. Workload and time to results are comparable for SNaPshot and direct sequencing. SNaPshot is flexible and easy to multiplex. The StripAssay is less time consuming for daily laboratory practice. SNaPshot is more flexible and slightly more sensitive than direct sequencing. The clinical evaluation showed comparable performances between direct sequencing and SNaPshot. The StripAssay is rapid and an extremely sensitive assay that could be considered when few tumor cells are available. However, found mutants should be confirmed to avoid risk of false positives.

The frequency and spectrum of K-ras mutations among Iraqi patients with sporadic colorectal carcinoma

“…[33] Analyzing several DNA samples taken from different parts of tumor might have provided useful The number in brackets under each country's name represents the total number of patients with K-ras mutations characterized by each study information relevant to the latter observation. Although we confirmed all cases with multiple mutations by repeating the testing, and mutant enriched PCR used in the current study is validated as highly sensitive and specific, [15,16] it may be argued that reaffirming results by another method may have been of added value. It is important to note here that the true clinical impact of multiple K-ras mutations is unknown, mainly due to the scarcity of such data.…”

“…K-ras gene point mutation detection was performed with the K-ras strip assay kit (Viennalab Diagnostic GmbH, Austria) which utilizes a mutant enriched PCR technique and reverse hybridization. [15,16] The DNA extracted was amplified with specific biotinylated primers using a thermocycler (ABI-USA) and the amplification program was Pre-PCR 94°C, following by 35 cycles of 94°C 1 min, 70°C 50 sec, 56°C 50 sec, 60°C 1 min, then by final extension at 60°C of 3 min. Following amplification the amplicons were reverse hybridized according to manufacturer's instructions, to readymade strips containing allele-specific oligonucleotide probes immobilized as an array of parallel lines to detect Statistical analysis utilized the Chi squared test and Mann Whitney U test when required.…”

Current companion diagnostics in advanced colorectal cancer; getting a bigger and better piece of the pie

“…Multiplex assays allow assessment of numerous genes within one reaction and have similar or better sensitivity to direct sequencing methods (58,117,118). They are cheaper than direct sequencing and are better able to deal with degraded or poor quality DNA.…”

Recommendations from the EGAPP Working Group: can testing of tumor tissue for mutations in EGFR pathway downstream effector genes in patients with metastatic colorectal cancer improve health outcomes by guiding decisions regarding anti-EGFR therapy?

“…In the only case in which there was poor agreement with sequencing results in this study, the corresponding assay has ceased to be available for commercial use. 30 In a recent study comparing SNaPshot and KRAS StripAssay with direct sequencing in detecting KRAS exon 2 mutations from 296 serially diluted samples, detection limits were 10% tumor cells with SNaPshot, 1% with the StripAssay, and 20% with direct sequencing. 30 Another recent study compared a Sequenom mass spectrometry genotyping assay with locked nucleic acid PCR sequencing, for KRAS and BRAF mutations, using 308 CRC samples that had originally been tested through standard sequencing.…”

“…Locked nucleic acid PCR detected 45%, and mass spectrometry 41%, more KRAS mutant samples than standard sequencing alone. 31 Although direct sequencing is considered the gold standard for KRAS mutation analysis, 30,32 it is less analytically sensitive (detects fewer mutated alleles in a heterogeneous mixture of mutated and unmutated cells) as compared with some real-time PCR-based KRAS assays. However, according to a 2009 College of American Pathologists (CAP) Perspectives on Emerging Technology report "the clinical relevance of a small percentage of cells with mutant KRAS has not been established. "…”