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SALSA MLPA Probemix P315 EGFR

Tumours, solid

Region: EGFR 7p11.2

General information: The SALSA MLPA Probemix P315 EGFR is a research use only (RUO) assay for the detection of deletions or duplications in human EGFR gene. This probemix can also be used to detect the presence of c.2573T>G=p.L858R and c.2369C>T=p.T790M point mutations.

The epidermal growth factor receptor (EGFR) is cell surface tyrosine kinase (TK) enzyme involved in controlling cell growth. Following binding of a ligand, EGFR stimulation leads to activation of downstream events including cell proliferation, differentiation, survival and DNA synthesis. Defects in EGFR is involved in the development of many cancers and undergoes various types of genetic alterations to gain oncogenic properties. Moreover, patients with tumours which have alterations in EGFR tend to have a more aggressive form of the disease, and expression levels of EGFR are highly predictive of clinical outcome for cancer patients.

The mechanisms for oncogenic conversion of EGFR in cancer include amplified copy number, structural rearrangements of the gene, and activating mutations that have all been detected in various malignancies. One of the most common deletions detected in tumours is the EGFR deletion variant III (EGFRvIII), which is an in-frame deletion of exons 2-7 leading to a ligand-independent receptor. In addition numerous other (exon) deletions and duplications are found in biopsies. EGFR mutations cluster in the kinase domain of EGFR (exons 18-21), and cause ligand-independent activation of the receptor, representing possible targets for therapeutic intervention. In this regard, certain somatic EGFR mutations as well as gene amplification in patients with non-small cell lung cancer (NSCLC) highly correlate with the clinical response to TK inhibitors. Two frequent mutations, c.2573T>G=p.L858R and c.2369C>T=p.T790M, are shown to be an important source of resistance to drugs acting on the TK domain of EGFR.

Probemix content:
The SALSA MLPA Probemix P315-C1 EGFR contains 47 MLPA probes with amplification products between 124 and 472 nt. This includes 28 probes for the EGFR gene, one upstream and two downstream flanking probes for the EGFR gene. Furthermore, it also contains two probes specific for the EGFR c.2573T>G=p.L858R and c.2369C>T=p.T790M point mutations which will only generate a signal when the mutations are present. In addition, 14 reference probes are included which target relatively copy number stable regions in various tumour types. Complete probe sequences and the identity of the genes detected by the reference probes are available in Table 2b and online (www.mlpa.com).

This probemix contains nine quality control fragments generating amplification products between 64 and 121 nucleotides (nt): four DNA Quantity fragments (Q-fragments), two DNA Denaturation fragments (D-fragments), one Benchmark fragment, and one chromosome X and one chromosome Y-specific fragment. More information on how to interpret observations on these control fragments can be found in the MLPA General Protocol and online at www.mlpa.com.

SALSA Binning DNA SD006: The SD006 Binning DNA provided with this probemix can be used as Binning DNA sample for binning of EGFR mutation-specific probes 22215-SP0449-L21566 for c.2573T>G=p.L858R mutation and probe 17162-SP0448-L21565 for c.2369C>T=p.T790M mutation. SD006 Binning DNA is a mixture of genomic DNA from healthy individuals and plasmid DNA that contains the target sequence detected by the above mentioned probes. Inclusion of one reaction with 5 μl SD006 Binning DNA in initial MLPA experiments is essential as it can be used to aid in data binning of the peak pattern using Coffalyser.Net software. Furthermore, Binning DNA should be included in the experiment whenever changes have been applied to the set-up of the capillary electrophoresis device (e.g. when capillaries have been renewed). Binning DNA should never be used as a reference sample in the MLPA data analysis, neither should it be used in quantification of mutation signals, as for this purpose true mutation/SNP positive patient samples or cell lines should be used. It is strongly advised to use test DNA sample and reference DNA samples extracted with the same method and derived from the same source of tissue. For further details, please consult the SD006 Binning DNA product description provided. This product is for research use only (RUO).

Sample DNA
Sample DNA developed for this product:

Order Items

Probemix

Item no.
Description
Technology
Price
P315-025R
SALSA MLPA Probemix P315 EGFR – 25 rxn
€ 243.00
P315-050R
SALSA MLPA Probemix P315 EGFR – 50 rxn
€ 486.00
P315-100R
SALSA MLPA Probemix P315 EGFR – 100 rxn
€ 972.00

Required Reagents (Sold Separately)

Item no.
Description
Technology
Price
EK1-FAM
SALSA MLPA EK1 reagent kit – 100 rxn – FAM
€ 300.00
EK1-Cy5
SALSA MLPA EK1 reagent kit – 100 rxn – Cy5
€ 300.00
EK5-FAM
SALSA MLPA EK5 reagent kit – 500 rxn – FAM
€ 1380.00
EK5-Cy5
SALSA MLPA EK5 reagent kit – 500 rxn – Cy5
€ 1380.00
EK20-FAM
SALSA MLPA EK20 reagent kit – 2000 rxn – FAM
€ 5295.00

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