Epidermal Growth Factor Receptor
EGFR and signaling with a scheme of EGFR Signaling
The epidermal growth factor receptor is a membrane-bound receptor that is among the tyrosine kinase family. EGFR is part of signaling pathways that control survival and cell division of cells. A mutation on the EGFR gene cause EGFR to be produced in amounts higher than normal in some types of cancer thus making the cancer cells grow faster. The signal transduction facilitated by EGFR is very complex. EGFR is served by two endogenous ligands that include transforming growth factor-alpha and EGF. Other ligands that have been classified include amphiregulin, betacellulin, epiregulin, epigene, and heparin-binding EGF-like growth factor. Each ligand seems to activate the EGFR similarly: binding of the ligand, receptor dimerization, receptor trans-autophosphorylation, and the recruitment of adaptors or signaling proteins. After a ligand binding, EGFR forms dimers with another EGFR. (David K. Lam, 2012)
Dimerization is followed by transmembrane domain rearrangement that leads to rearrangement of juxta-membrane segments and then autophosphorylation occurs resulting in a series of intracellular signaling activities which involve activation of signal transducer and activator of transcription, protein kinase C, and Ras/Raf/mitogen-activated protein kinase. The signaling pathway then mediates cell survival, proliferation, invasion, angiogenesis, and metastasis (Ping Wee, 2017). The propagation of signals through the various complex pathways then induces the expression of new genes. The up-regulation of EGFR is mediated by various mechanisms such as truncations to its extracellular domain and common mutations. The expression of EGFR in normal cells is estimated to around 40,000 to 100,000. The gene is mapped in the short am of chromosome 7 q22. EGF addition to HeLa cells has been shown to activate EGFR to cause phosphorylation of 2244 proteins at 6600 different sites. The truncation and mutation of EGFR can impart it with ligand-independent signaling, which causes the rise in various pro-oncogenic processes that involves chronic cell cycle proliferation. (NIH)
EGFR mutation testing methods
Detection of EGFR mutations is normally done on DNA samples of tumor tissues obtained by surgical resection or biopsy using gene sequencing. The samples to be analyzed using direct sequencing are normally in form of formalin-fixed paraffin-embedded diagnostic blocks. Gene sequencing has been considered the gold standard for EGFR mutation testing for some time, however, due to a few disadvantages such as low sensitivity and high risk of missing EGFR mutations in positive individuals, other alternative methods of mutation testing have been introduced. (Cappuzzo, 2014)The use of high-performance liquid chromatography to analyze frozen tissue samples has shown consistent results as those obtained by direct sequencing. Another method that has been shown to have higher sensitivity and specificity when compared to sequencing is high-resolution melting analysis (HRMA). (Cappuzzo, 2014)
A study performed by Querings et al examined an alternative next-generation sequencing called massively parallel sequencing which yielded a 100% success rate in detecting low-frequency EGFR mutations. The other effective method is pyrosequencing which is a non-electrophoretic sequencing technology that employs luminometric detection which was identified to have an 89% success rate in detecting low-frequency mutations. Polymerase chain reaction-based methods have also be used in detecting EGFR mutation specifically to detect exon 19 deletions. There is also the Amplification refractory mutation system which is a method that helps to discriminate between mutated and wild-type DNA through selective amplification of targeted mutation-containing sequences. (Gillian Ellison, 2013)
Mutations in Lung cancer.
Several mutations can contribute to the development of non-small cell lung cancer. The mutations can either be a somatic mutation that is acquired or hereditary germline mutation. The different types of mutations in lung cancer include TP53 gene mutation. The TP53 gene is responsible for tumor protein p53 production. The p53 protein acts as a tumor suppressor and monitors cells for DNA damage. Another mutation in the KRAS gene mutation found in 30% of all NSCLCs and is common among individuals who smoke. EGFR mutation occurs in about 23% of NSCLCs and is commonly associated with nonsmokers and women. (Watson, 2016)The anaplastic lymphoma kinase (ALK) mutation occurs in 5% of NSCLC tumors and is common among younger people and nonsmokers. The MET and METex14 are observed in 5 percent of all NSCLCs and cancer tend to be more aggressive. There are also BRAF mutations that occur in about 3 to 4 percent NSCLCs. And finally, there are the ROS1 mutations that affect 1 and 2 percent of NSCLCs and are found mainly in younger people who have never smoked.
Inhibition of mutated epidermal growth factor EGFR is one of the most successful cancer-targeted therapy. Even though the treatment has benefited most people with activating EGFR, resistance is eventually acquired by almost all who benefited. About 50 percent of cases of acquired resistance are normally due to a secondary T790M mutation that occurs in exon 20 of the EGFR gene. However, most of the mechanism that leads to resistance are not well understood. EGFR is inhibited by a group of small molecule inhibitors that inhibits the tyrosine kinase domain of EGFR. Despite the treatment, a majority developed resistance after treatment with the first generation EGFR TK1, typically in less than a year among patients with activating mutations. A summary of some of the mechanisms of resistance to first-generation EGFR TK1s can be illustrated in the diagram below. (Tianli Zhang, 2019)
The primary resistance involves the EGFR somatic mutations such as the L858R and exon 19 deletions. The EGFR germline polymorphism has also been associated with primary resistance alongside other genetic polymorphisms. Acquired resistance includes secondary EGFR mutations i.e. T790M mutations, gene copy alterations of alternative pathways, mutations in downstream effector molecules of EGFR, epigenetics, and other mechanisms. (Tianli Zhang, 2019)
Sensitizing mutations in EGFR are associated with a positive response to anti-EGFR-targeted therapy. Exon 21L858R substitutions and exon 19 deletions are the most common mutations that account for about 90 percent of mutations in NSCLC. These mutations classically increase the sensitivity to tyrosine kinase inhibitors. The uncommon EGFR mutations of which S768I, G719X, L861Q, complex mutations, and exon 20 insertions are the most common. The uncommon mutations that are sensitive to first-generation TK1s are the S7681, L861Q, G719X, point mutations, and those mutations that exist within the complex mutation. The uncommon EGFR mutations indicate a variation in efficacy to EGFR-targeted drugs depending on the molecular alterations that occur within exon 18-21.