The EGFR FISH probe set is designed to measure the copy number of the human EGFR gene located on chromosome band 7p11.2. The kit contains two differentially labeled probes: A Locus Specific Probe (LSP) covers a chromosomal region which includes the entire EGFR gene, along with some upstream (5’) and a small amount of downstream (3’) nontranslated genomic sequences. The other probe, CCP7, is derived from chromosome 7-specific alpha satellite DNA and is designed to serve as a control to determine the number of chromosome 7 copies per cell.

Fluorescence in situ hybridization (FISH) is a powerful technique developed to detect presence or absence, location, integrity and amount of genomic sequences in tissue samples or cells.^1-3 

Epidermal Growth Factor Receptor (EGFR – also called ERBB, ERBB1, HER1, PIG61, mENA) is a membrane protein found in all vertebrate animal species. In humans, it is a member of a subfamily of four closely related receptor tyrosine kinases, together with HER2/c-neu (ErbB-2), HER3 (ErbB-3) und HER4 (ErbB-4). EGFR is a single-pass transmembrane protein with an intracellular (cytoplasmic) intrinsic kinase domain and ATP binding site. Activation of the receptor is initiated by growth factor interaction with the extracellular ligand binding domain, which triggers receptor dimerization, autophosphorylation and recruitment of mediators of several signal transduction pathways affecting cell growth, survival and differentiation.^4,5

Epidermal growth factor (EGF), one of the earliest known polypeptide growth factors, and the more widely expressed transforming growth factor-alpha (TGF-α) are the most thoroughly studied EGFR ligands, but several other factors can also activate this receptor, such as Heparin-binding EGF-like growth factor (HB-EGF), amphiregulin, betacellulin, epigen and epiregulin.^6,7

EGFR and Cancer 

Normally expressed in most human cells and tissues, EGFR has been found activated in many tumor types including lung, colorectal, head and neck, breast, cervical, oral, pancreatic and other tumors, and gliomas.^8-15 These observations have prompted development of targeted therapeutics aimed at thwarting oncogenic signaling through EGFR. Among them, small molecule tyrosine kinase inhibitors for lung cancer treatment (gefitinib, erlotinib) and monoclonal antibodies for colon cancer therapy (cetuximab, panitumumab) have advanced the furthest in clinical development, but many other promising agents are in trials for various malignancies (e.g. zalutumumab, nimotuzumab, matuzumab, lapatinib, afatinib, dacomitinib, XL647, AP26113, CO-1686).

Although remarkable, treatment successes in first-generation compound trials were limited to small patient subgroups, in many cases later found to harbor characteristic kinase domain or other point mutations. Moreover, most patients eventually developed drug resistance. This has on the one hand led to the development of newer drug generations.¹⁶ On the other hand it has generated a vigorous and still ongoing debate as to whether mutation analysis, copy number testing or other (e.g. proteomic) methods will result in identification of the best predictors of response to EGFR targeted therapy.^17-21 A high degree of intratumor heterogeneity might turn out to be one reason for the controversial clinical data existing regarding responsiveness.²² On the whole, however, high EGFR copy number as measured by FISH can be a robust marker of poor prognosis, and it is now broadly acknowledged that in many disease subtypes better outcomes can be expected in EGFR FISH positive tumors.^23-25

While active EGFR can also be detected by immunohistochemistry, measuring EGFR expression by IHC has not resulted in a reproducible and reliable predictor in clinical practice, whereas gene copy number evaluation by FISH has proven itself as one of the most effective markers for sensitivity to EGFR inhibitor therapy.^26-29

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