therascreen RAS Extension Pyro Kit

LOB and LOD were determined according to the recommendations in Clinical and Laboratory Standards Institute (CLSI) Guideline EP 17A “Protocol for determination of limits of detection and limits of quantitation; approved guideline”, and EP17-A2 “Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures; Approved Guideline—Second Edition”. LOD of the mutations covered by the therascreen RAS Extension Pyro Kit are ranging from 1.8% to 8.8%.  

Linearity

Linearity was determined according to the CLSI Guideline EP6-A2 “Evaluation of the linearity of quantitative measurement procedures: a statistical approach; approved guideline”. All assays were linear within an allowable nonlinearity of 5 % units in the tested range of 5 to 50% mutation level.

Precision

The precision data allows the determination of the total variability of the assays and was obtained at 3 different levels by analysis of the above mentioned plasmid mixtures with 3 replicates each.

• Repeatability (intra-assay and inter-batch variability) was calculated based on the data for determination of linearity (3 runs on the same day using varying lots of the therascreen RAS Extension Pyro Kit).
• Intermediate precision (intra laboratory variability) was determined in 3 runs within one laboratory on 3 different days with varying operators, PyroMark Q24 instruments, and lots of the therascreen RAS Extension Pyro Kit.
• Reproducibility (inter-laboratory variability) was calculated from 2 runs each in two independent laboratories and using varying lots of the therascreen RAS Extension Pyro Kit.

Precision results are described in the therascreen RAS Extension Pyro Kit handbook.

The therascreen RAS Extension Pyro Kit is used for quantitative measurements of mutations in exons 3 and 4 of the human KRAS oncogene, and exons 2, 3, and 4 of the human NRAS oncogene using Pyrosequencing technology on the PyroMark Q24 System. The RAS genes encode proteins that play a critical role in the EGFR signaling cascade. Mutations in the RAS genes can affect how the proteins stimulate these downstream pathways.

The following mutations are detected:

For a general overview of Pyrosequencing technology, please visit our Pyrosequencing resource center

The therascreen RAS Extension Pyro Kit consists of 8 assays covering the different regions of interest (see Figure 1. Assays of the therascreen RAS Extension Pyro Kit and Figure 2. Pyrogram trace obtained after analysis of a sample with a wild-type genotype with the KRAS 146 assay.). The regions are amplified separately by PCR and sequenced through the defined region. Sequences surrounding the defined positions serve as normalization and reference peaks for quantification and quality assessment of the analysis.

After PCR using primers targeting the different codons, the amplicons are immobilized on Streptavidin Sepharose High Performance beads. Single-stranded DNA is prepared, and the corresponding sequencing primers anneal to the DNA. The samples are then analyzed on the PyroMark Q24 System using a run setup file and a run file. The RAS Extension Plug-in Report should be used to analyze the run. This report ensures that the correct limit of detection (LOD) values are used and different sequences to analyze are automatically used to detect all mutations (see Figure 3. RAS Extension Plug-In Report menu and Figure 4. RAS Extension Plug-In Report). However, the run can also be analyzed using the analysis tool integral to the PyroMark Q24 System. The "Sequence to Analyze" can be then adjusted for detection of rare mutations after the run.

The therascreen RAS Extension Pyro Kit is an in vitro diagnostic test based on Pyrosequencing^® technology for the quantitative detection of mutations in codons 59, 61, 117, and 146 of the human KRAS oncogene and codons 12, 13, 59, 61, 117, and 146 of the human NRAS oncogene, using DNA extracted from formalin-fixed paraffin-embedded (FFPE) metastatic colorectal cancer (mCRC) human tissue.

The therascreen RAS Extension Pyro Kit is intended to aid in the identification of mCRC patients more likely to benefit from anti-EGFR therapies such as cetuximab and panitumumab.