therascreen GIST RapidScreen Pyro Kit

For quantitative detection of mutations in the human KIT and PDGFRA genes

Features

Reliable and clinically relevant results on mutation status within hours
Detection of insertion in exon 9 of KIT related to dosage of imatinib
Detection of mutation in exon 18 of PDGFRA related to imatinib resistance
Developed together with GIST reference center CIO in Cologne, Germany

Product Details

The therascreen GIST RapidScreen Pyro Kit is a molecular detection kit for rapid quantitative detection of mutations in the human KIT and PDGFRA genes. The kit contains enzymes, primers, and reagents for amplification of the genes plus buffers, primers, and reagents for detection and quantification of mutations in real time using Pyrosequencing technology on the PyroMark Q24 System.

Performance

The limit of blank (LOB) and limit of detection (LOD) have been determined for a number of mutations using mixtures of plasmids (see table "LOB and LOD determined for specific mutations"). LOB and LOD were determined according to the recommendations in the Clinical and Laboratory Standards Institute (CLSI) Guideline EP17 A “Protocol for determination of limits of detection and limits of quantitation; approved guideline”. Alpha- and beta-errors (false positive and false negative, respectively) were set to 5%. LOD for some rare deletions in PDGFRA exon 18 were determined by adding 3 standard deviations of blank measurements to the LOB value. The LOD values were set at least 3 % units above the LOB value.
LOB values represent the measured frequency obtained with a wild-type sample. LOD values represent the lowest signal (measured frequency) that can be regarded as positive for the respective mutation.

LOB and LOD determined for specific mutations
Nucleic acid substitution	Amino acid substitution	LOB (% units)	LOD (% units)	Cosmic ID* (v58)
KIT exon 9
1509_1510insGCCTAT	Y503_F504insAY	1.9	4.9	1326
PDGFRA exon 18
2525A>T	D842V	0.6	3.6	736
2524G>T	D842Y^†	0.6	3.6	12396
2524_2535 del12 or^‡ 2526_2537 del12	D842_H845del or^‡ I843_D846del^†	2.2	5.2	737 or^‡ 96892
2527_2538 del12	I843_D846del†	3.0	5.0	12400
2528_2539 del12	I843_S847>T	4.2	7.2	12407
2530_2541 del12	M844_S847del	3.2	6.2^§	12402
2524_2532 del9	D842_M844del	1.5	4.5	12401
2524_2526 delGAC	D842del	0.9	3.9^§	12406
2526_2538 >G^¶	D842_D846>E	0.3	3.3^§	12408
2524_2526 GAC>TAT	D842Y^†	0.9	3.9^§	12397

* From the Catalogue of Somatic Mutations in Cancer, available online at the Sanger Institute at www.sanger.ac.uk/genetics/CGP/cosmic.
^† The mutations 2524G>T and 2524_2526 GAC>TAT, and 2526_2537 del12 and 2527_2538 del12 result in the same amino acid change, respectively.
^‡ The mutations 2524_2535del12 and 2526_2537del12 result in the same nucleic acid change.
^§ The LOD for these deletions in PDGFRA exon 18 were determined by adding 3 standard deviations of blank measurements to the LOB value.
^¶ The mutation 2526_2538 >G cannot be analyzed in the AQ mode of the PyroMark Q24 Software.

See the kit handbook for additional performance data.

Principle

The therascreen GIST RapidScreen Pyro Kit is used for quantitative measurements of mutations in KIT exon 9 and PDGFRA exon 18. Detection of mutations in KIT exon 9 allow the use of the appropriate dose of imatinib and detection of mutations in PDGFRA exon 18 help to exclude less sensitive or resistant genotypes (1–3).
The kit consists of two assays: one for detecting mutations in KIT exon 9 and the other for detecting mutations in PDGFRA exon 18. The two 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.
The product consists of a PCR primer mix and a sequencing primer for each assay. The primers are delivered in solution. Each vial contains 32 µl of each primer or primer mix.

Cited references

The ESMO/European Sarcoma Network Working Group (2012) Gastrointestinal stromal tumors: ESMO clinical practice guidelines for diagnosis, treatment and follow-up. Ann. Oncol. 23 (Supplement 7), vii49.
Gastrointestinal Stromal Tumor Meta-Analysis Group (MetaGIST) (2010) Comparison of two doses of imatinib for the treatment of unresectable or metastatic gastrointestinal stromal tumors: A meta-analysis of 1,640 patients. J. Clin. Oncol. 28, 1247.
Joensuu, H. (2006) Gastrointestinal stromal tumor (GIST). Ann. Oncol. 17 (Supplement 10), x280.

Procedure

After PCR using primers targeting KIT exon 9 and PDGFRA exon 18, 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 using assay setup files and a run file.
It is recommended to use the GIST RapidScreen Plug-in Report to analyze the run. The GIST RapidScreen Plug-in Report can be obtained by email from pyro.plugin@qiagen.com. However, the run can also be analyzed using the analysis tool integral to the PyroMark Q24 system. The “Sequence to Analyze” can be adjusted for detection of rare mutations after the run.

Applications

The therascreen GIST RapidScreen Pyro Kit is an in vitro nucleic acid sequence-based detection test based on Pyrosequencing technology for the quantitative detection of mutations in exon 9 of the human KIT gene and in exon 18 of the human PDGFRA gene in genomic DNA derived from human tissue samples.
The therascreen GIST RapidScreen Pyro Kit is intended to provide clinicians with information to aid the management of patients diagnosed with gastrointestinal stromal tumor (GIST) more likely to benefit from drugs targeting signaling pathways, such as imatinib. For in vitro diagnostic use.
This product is not intended for use with samples from lung tissue.