EGFR testing for laboratories

The only FDA-approved assay for the use with GILOTRIF, IRESSA or XEGAFRI

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therascreen EGFR RGQ PCR Kit (therascreen EGFR test) is the only FDA-approved test for the selection of patients with non-small cell lung cancer (NSCLC) for whom GILOTRIF (afatinib), IRESSA (gefitinib) or XEGAFRI (rociletinib), EGFR tyrosine kinase inhibitors (TKIs), are indicated. This rapid and sensitive assay is part of an easy and reliable system with automated result reporting. The kit enables you to identify 21 mutations, including the most prevalent activating mutations, exon 19 deletions and exon 21 L858R substitution mutations, and the most important resistance mutation, T790M

Principle
Procedure
Application
Principle

The therascreen EGFR RGQ PCR Kit is a real-time PCR test for the qualitative detection of exon 19 deletions and exon 21 (L858R) substitution mutations of the epidermal growth factor receptor (EGFR) gene in DNA derived from formalin-fixed paraffin-embedded (FFPE) non-small cell lung cancer (NSCLC) tumor tissue. The test is intended to be used to select patients with NSCLC for whom GILOTRIF (afatinib) or IRESSA (gefitinib) EGFR tyrosine kinase inhibitors (TKIs) are indicated. Safety and efficacy of GILOTRIF (afatinib) and IRESSA (gefitinib) have not been established in the patients whose tumors have L861Q, G719X, S768I, exon 20 insertions, and T790M mutations, which are also detected by the therascreen EGFR RGQ PCR Kit.

ARMS

Allele- or mutation-specific amplification is achieved by ARMS (Amplification Refractory Mutation System). ARMS primers preferentially anneal with DNA containing the mutation and allow Taq DNA polymerase to initiate PCR, effectively distinguishing between a match and a mismatch at the 3' end of a PCR primer. Specific mutated sequences are selectively amplified, even in samples where the majority of the sequences do not carry the mutation. When the primer is fully matched, the amplification proceeds with full efficiency. When the 3' base is mismatched, only low-level background amplification occurs. 

Scorpions

Detection of amplification is performed using Scorpions. Scorpions are bi-functional molecules containing a PCR primer covalently linked to a probe. The technology uses a fluorescence-based method to indicate the presence of the mutation. The Scorpion primer hybridizes with a DNA sequence upstream of the target region. The primer is then extended by Taq DNA polymerase and the target region is copied. The newly copied region is complementary to the probe region of the Scorpion. Following a temperature increase within the real-time PCR cycler, the extended Scorpions primer denatures. When the solution cools, the Scorpions probe self hybridizes. The fluorophore is separated from the quencher and a fluorescence signal is generated.

 

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Procedure

Extract DNA samples from FFPE NSCLC tumor tissue collected from NSCLC patients using the QIAamp DSP DNA FFPE Tissue Kit, which has been validated for use with the therascreen EGFR RGQ PCR Kit. The therascreen EGFR RGQ PCR Kit uses a two-step procedure. The first step is performance of the control assay to assess the total amplifiable DNA in a sample. The second step is to test with the mutation assays in order to detect the presence or absence of EGFR mutations.

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Application

The therascreen EGFR RGQ PCR Kit enables the detection of 21 mutations of the human EGFR gene using DNA extracted from FFPE NSCLC tumor tissue samples. The therascreen EGFR RGQ PCR Kit is only intended to discriminate between EGFR mutation-negative (wild-type) and EGFR mutant tumors. EGFR mutations detected by the therascreen EGFR RGQ PCR Kit include:

EGFR mutations detected using the therascreen EGFR RGQ PCR Kit

Mutation

Exon

Base change

Cosmic ID

Deletions


19

2238_2255del18

6220

2235_2249del15

6223

2236_2250del15

6225

2239_2253del15

6254

2239_2256del18

6255

2240_2254del15

12369

2240_2257del18

12370

2239_2248TTAAGAGAAG>C

12382

2239_2251>C

12383

2237_2255>T

12384

2239_2258>CA

12387

2238_2252>GCA

12419

2238_2248>GC

12422

2235_2252>AAT

13551

L858R

21

2573T>G

6224

T790M*

20

2369C>T

6240

L861Q*

21

2582T>A

6213

G719A*

18

2156G>C

6239

S768I*

20

2303G>T

6241

Insertions*

20

2319_2320insCAC

12377

2310_2311insGGT

12378

*Safety and efficacy of GILOTRIF (afatinib), IRESSA (gefitinib) and XEGAFRI (rociletinib) has not been established for these mutations.

 
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