therascreenEGFR RGQ PCR Kit

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) areindicated. Safety and efficacy of GILOTRIF (afatinib) has not been established in patients whose tumors have exon 20 insertions and T790M mutations. Safety and efficacy of IRESSA (gefitinib) has not been established in patients whose tumors have L861Q, G719X, S768I, exon 20 insertions and T790M mutations. These EGFR mutations 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.

Extract DNA samples fromFFPE NSCLCtumor tissue collected fromNSCLC patients using the QIAamp DSP DNA FFPE Tissue Kit, which has been validated for use with the therascreenEGFR RGQ PCR Kit. The therascreenEGFR 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 ofEGFR mutations.

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

*Safety and efficacy of IRESSA (gefitinib) has not been established for these mutations.

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