Epidermal growth factor receptor (EGFR) encodes a transmembrane glycoprotein. This protein is a member of the protein kinase superfamily, which consists of EGFR (ErbB1/HER1), HER2 (ErbB2), HER3 (ErbB3) and HER4 (ErbB4). All family members contain an extracellular ligand-binding domain, a single membrane-spanning region, a juxtamembrane nuclear localization signal, and a cytoplasmic tyrosine kinase domain. They are collectively called as HER receptors and are ubiquitously expressed in various cell types, but primarily in those of epithelial, mesenchymal and neuronal origin. Under homeostatic conditions, receptor activation is tightly regulated by the availability of ligands, which together form the epidermal growth factor (EGF) family [Article:11252954]. From those ligands, EGF, transforming growth factor alpha and amphiregulin bind specifically to EGFR [Article:15864276]. Binding of the EGFR or other family members to a ligand induces receptor dimerization and tyrosine autophosphorylation and leads to cell proliferation. The EGFR involvement in carcinogenesis has been well established and mutations in EGFR can be utilized as predictive markers in the treatment of cancer.
EGFR Gene, Molecular Structure and Function
EGFR maps on chromosome 7p11.2, it covers 188.3 kb, from 55,086,725 to 55,275,031, on the positive strand. EGFR is composed of 28 exons and encodes a protein of 1210 amino acids (ENST00000275493, Ensembl v69) [Article:11752248]. Multiple alternatively spliced transcript variants that encode different protein isoforms have been found [Article:16925834].
EGRF activation by binding of growth factor leads to the autophosphorylation of the intracellular tyrosine kinase domain and results in the formation of receptor homodimers or heterodimers with other HER family members, and the tyrosine phosphorylated residues act as a docking site for various adapter molecules, and resulting in the activation of the downstream signaling pathways [Articles:18375904, 12648464], driving different biological processes including cell cycle progression and differentiation, increased cell invasiveness, apoptosis and angiogenesis [Articles:16014887, 11129168]. Thus, overexpression of EGFR is believed to have a critical role in tumor progression [Articles:16014887, 11129168, 16377102].
The principal cause of cancer-related mortality is lung cancer, and non-small cell lung cancer (NSCLC) constitutes almost 80% of all lung cases. NSCLC is arisen from lung epithelial cells, and comprises diverse histological subtypes including adenocarcinoma, bronchioloalveolar, squamous, anaplastic and large-cell carcinomas, about half of the NSCLC patients manifest advance disease at the time of diagnosis thus making the treatment difficult [Article:18287387]. Various oncogenic mechanisms, including EGFR gene mutations, increased EGFR copy number and EGFR protein overexpression may impair the regulation of tyrosine kinase activity of EGFR in tumor cells [Articles:18337605, 20388064] and may result in increased malignant cell survival, proliferation, invasion and metastasis [Article:19138950]. The current approach is that patients with specific types and stages of cancer should be treated according to standardized, predetermined protocols [Article:22594511]. However, understanding the molecular genesis of NSCLC and advances in the field of pharmacogenomics can lead to a rational use of targeted therapies.
EGFR as cancer drug target
EGFR has been linked to the growth of many human epithelial malignancies, including NSCLC, metastatic colorectal cancer (CRC), head and neck squamous-cell carcinoma (HNSCC), and pancreatic cancer [Articles:16377102, 20551942, 18681783]. Intensive laboratory and clinical research have facilitated development of EGFR inhibitors. There are two main types of EGFR inhibitors; tyrosine kinase inhibitors and monoclonal antibodies against EGFR (http://pharmgkb.org/pathway/PA162356267).
Tyrosine Kinase Inhibitors (TKIs): TKIs are synthetic molecules that block ligand-induced receptor autophosphorylation by binding to the ATP-binding pocket of the intracellular tyrosine kinase domain and disrupting tyrosine kinase activity, thus eliminating intracellular downstream signaling [Articles:18375904, 12648464].
Gefitinib and erlotinib are specific for EGFR, whereas afatinib, lapatinib and neratinib inhibits EGFR (HER1) and HER2; pelitinib inhibits EGFR, HER2 and HER4; and vandetanib inhibits EGFR, vascular endothelial growth factor receptor (VEGFR) and the RET-tyrosine kinases [Article:20551942].
The FDA approved gefitinib through an accelerated process in May 2003 as monotherapy for the treatment advanced NSCLC patients after failure of both platinum-based and docetaxel chemotherapies. As a condition of accelerated approval, the FDA required demonstration of a survival benefit in a subsequent clinical trial. Three large, prospective studies showed no improvement in overall survival [Articles:14990632, 14990633, 16257339], therefore the original FDA approval for gefitinib was modified. Currently gefitinib is indicated as monotherapy for the continued treatment of advanced NSCLC patients after failure of both platinum-based and docetaxel chemotherapies who are benefiting or have benefited from gefitinib (http://dailymed.nlm.nih.gov/dailymed/).
Erlotinib monotherapy is indicated for the treatment of advanced NSCLC patients after failure of prior chemotherapy regimen. FDA also approved erlotinib in combination with gemcitabine for advanced pancreatic cancer patients who have not received previous chemotherapy (http://dailymed.nlm.nih.gov/dailymed/).
Previously, treatment outcomes of erlotinib or gefitinib were studied in unselected patients presenting conflicting results depending on the type of patient population enrolled in each study. However, the discovery that response to erlotinib or gefitinib is associated with the presence of activating somatic EGFR mutations in NSCLC has led to the design of clinical trials in which patients were selected on the basis of the EGFR mutational status [Articles:22594511, 23022519]. This pharmacogenetic approach and its results will be discussed in detail below.
Other TKIs (lapatinib, neratinib, pelitinib and vandetanib) were either approved or in clinical trial phases for cancers other than NSCLC (http://dailymed.nlm.nih.gov/dailymed/). Several clinical trials are continuing for afatinib and preliminary result of one of these trials will be discussed in the context of treatment of advanced NSCLC harboring activating somatic EGFR mutations.
Monoclonal antibodies: Cetuximab and panitumumab are monoclonal antibodies that specifically target the extracellular domain of EGFR. Cetuximab functions by blocking endogenous ligand binding to the extracellular domain of EGFR and enhances receptor internalization and degradation [Articles:6298788, 11255078]. Cetuximab and panitumumab were approved for the treatment of patients, other than NSCLC, with EGFR-expressing metastatic CRC refractory to chemotherapy [Articles:18316547, 19339720, 18003960]. Cetuximab was also approved for the treatment of advanced HNSCC in combination with radiation therapy [Articles:18784101, 16467544]. Since cetuximab and panitumumab block extracellular domain of EGFR, not TK domain, activating mutations might not affect treatment outcome.
Genetic variation of EGFR: Somatic mutations & germline SNPs
COSMIC database, designed to store somatic mutation information and related details of human cancers was used to explore EGFR somatic mutations (release v62, date of access: 12/03/2012, http://www.sanger.ac.uk/cosmic) [Article:20952405].
Out of 68,986 unique samples deposited in the COSMIC database for EGFR (partial or full sequence and genotype data) including all cancers examined, 13,201 (19.1%) samples had somatic mutations and about 1.3% of all samples has more than one mutation. There are 842 unique location entries for somatic EGFR mutations. Among the mutation bearing patients, six of the mutations have a frequency >= 1% and five has 0.1%-1% frequency, remaining somatic mutations were spread out along EGFR, mostly missense substitutions but there are insertions and deletions as well. All six common somatic mutations (>= 1%) constitutes ~93% of all mutations and are in the tyrosine kinase domain (between 712 and 968 amino acids, exon 18-24) of EGFR (Table 1). The most common one is exon 19 (codon 729-761) mutations, essentially it is not a simple mutation, rather collection of different deletions and a few missense substitutions concentrated on codons 744-753 of exon 19, the most frequent one of this group is E746_A750del mutation. Exon 19 mutations comprise 48.3% of all mutations.
The second common mutation is L858R (rs121434568, T-to-G change at middle base of the codon) and comprises 36.2% of all mutations. Other missense mutations were also observed on this codon in different base(s) as monoallelic or biallelic mutation combinations (L858K, L858M, L858Q, L858R and L858L) in one or few subjects. The third common mutation, T790M (rs121434569) is detected in 3.8% of all mutations. The forth common is exon 20 mutations, a group of different insertions were concentrated at codons 763-774 and this group comprises 2.3% of all mutations. The fifth common one is observed at codon 719; mutation at the first base of codon 719 is in dbSNP as rs28929495 (G719S/G719C) and second base mutations give rise to G719A or G719D; all 719 codon mutations comprises 1.6% of all mutations. The last common mutation, L861Q (rs121913444) comprises ~1% of all mutations. L861R and L861V mutations were also observed on this codon in one or few subjects. The five rare mutations (0.1%-1% frequency) are A289V, G598V, E709K, S768I and L833V; and totaling ~1% of all mutations (Table 1).
Table 1: Incidence of the Common (1%) and Rare (0.1%-1% ) Specific Somatic Mutations of EGFR.
|Exon 19 mutations: Collection of different deletions and a few missense substitutions, the most frequent one is E746_A750del||48.3%|
|L858R (rs121434568). Other missense mutations were also observed on this codon in extremely low frequency||36.2%|
|Exon 20 mutations: A group of different insertions concentrated at codons 763-774||2.3%|
|Codon 719 mutation: Mutation at the first base of codon G719S or G719C (rs28929495), 0.82%; Mutation at the second base of codon G719A or G719D, 0.77% ||1.6%|
|L861Q (rs121913444). L861R and L861V mutations were also observed on this codon in extremely low frequency ||~1.0%|
|A289V, G598V, E709K, S768I and L833V: combined incidence|| ~1.0%|
|*Incidences are derived from COSMIC database. 68,986 unique samples are deposited for EGFR of which 69% of are from lung cancer tissues||.|
TK domain of EGFR (exon 18-21) was sequenced or assayed by TaqMan probes for known mutations in other than lung cancers. Although EGFR somatic mutations were not observed in many cancer tissues [Articles:15741570, 16199108, 16353180, 22252115, 22426987], when systematic approaches with more samples were collected as in COSMIC database [Article:20952405], mutations were observed in other cancers tissues. For the EGFR mutations in COSMIC database, the majority of the samples (69% of 68,986 unique samples) are derived from lung cancer tissues and remaining samples are derived from 38 different cancer tissues. EGFR mutations are observed at 7.4% of the lung cancer samples and 1-2% of salivary gland, eye, peritoneum, upper aerodigestive tract, adrenal gland, and thyroid cancer tissues. Of the 39 cancer tissue results deposited, 22 of them have EGFR mutations ranging from 0.1% (pancreas, hematopoietic and stomach tissues) to 7.4% (lung) of their respective tissues.
In a recent study, whole exome and genome sequences of 183 lung adenocarcinoma tumor/normal DNA pairs were analyzed and EGFR mutations were observed at 17.5% of patients with a few of them having more than one mutation. The L858R (rs121434568) and exon 19 deletions constituted half of the EGFR mutations [Article:22980975]. In contrast, whole exome sequencing of 31 NSCLC revealed a L858R mutation in only one patient (3.2%) [Article:22510280]. Several somatic mutations were also observed in genes other than EGFR [Articles:22980975, 22510280].
L858R (rs121434568) and exon 19 deletions: EGFR mutations that lead to increased response to epidermal growth factors are called activating mutations, thus having these mutations produce a more significant and persistent activation of intracellular signaling pathways, resulting in increased cell proliferation. On the other hand, lower concentrations of TKIs are required to inhibit TK phosphorylation, because the mutant receptor has reduced ATP affinity that accounts for increased sensitive to drugs as compared with wild type EGFR [Articles:16014887, 15118073, 19147750]. EGFR kinase domain mutations that are clustered around the ATP-binding pocket of the enzyme exon 19 mutations, L858R, G719X (G719C, G719S and G719A) and L861Q increase the kinase activity of EGFR therefore they are activating mutations [Articles:15118073, 19147750]. There are many rare mutations in this region that their functionalities have not been determined. The L858R and exon 19 mutations constitutes ~84.5% of COSMIC, 86.7% [Article:18670300] and 90.9% [Article:17888036] of all mutations, therefore many studies utilize these two mutations in their analysis.
Although prospective studies did not demonstrate increased overall survival [Articles:14990632, 14990633, 16257339] as first-line treatment for NSCLC, several trials have confirmed their clinical usefulness as second- or third-line therapy in advanced NSCLC based on longer progression free survival (PFS) and lower toxicity obtained with TKIs as compared to standard therapy [Articles:16014882, 19027483]. However, clinical responses to both erlotinib and gefitinib differ among NSCLC patients, approximately 10% of patients had clinical responses when treated with TKIs [Articles:14990632, 14990633, 16257339, 16014882, 19027483]. Sequencing of the EGFR in tumor samples from these responders showed somatic gain-of-function (e.g. activating) mutations and this guide to the new clinical trials or retrospective analysis in which patients were chosen depending on the activating EGFR mutational status [Articles:22594511, 23022519].
Clinical responses for NSCLC patients harboring EGFR mutations were evaluated who are treated with TKIs in retrospective or prospective studies. Patients with an activating somatic EGFR mutation had significantly increased response rate (RR)[Articles:22740981, 22370314, 18349398, 17387341, 16956694, 20038723, 16204011, 17047654, 17473659, 17106442, 15897572, 15897572, 16203769, 17317677, 16865253, 16115929, 17285735, 15118073, 22982650, 17285735] and longer progression-free survival (PFS) [Articles:22370314, 18349398, 17387341, 16956694, 22982650, 21969500, 17429313, 17192902, 15897572, 16043828, 17047654, 20038723, 22215752, 22760226] time compared to patients who have no somatic mutation when treated with erlotinib or gefitinib. Although none of the prospective studies reported a statistical overall survival (OS) advantages, in a few relatively small studies analyzed mutational status retrospectively and suggested that OS was increased in mutation harboring East Asian NSCLC patients when treated with gefitinib [Articles:16956694, 17429313, 17192902, 16043828, 16865253, 17106442, 22370314, 22982650]. EGFR mutations were present in most cases of NSCLC patients who responded well to TKIs, yet approximately 10-20% of patients who do show a partial response to gefitinib do not have identifiable EGFR mutations, indicating that EGFR mutations are not the sole determinants of TKI response [Article:17318210]. Most studies presented their results according to activating somatic EGFR mutation status, regardless of the mutation type, but all or majority of the mutations were either rs121434568 (L858R) or exon 19 deletion(s).
Having better clinical output with TKIs in patients with activating EGFR mutations led to new clinical trial design. In prospective phase III randomized trials comparing TKIs and chemotherapy as first-line therapy in patients with advanced NSCLC harbouring activating EGFR mutations, erlotinib [Article:21783417] gefitinib [Articles:20022809, 20573926] and afatinib treatment arms had significantly increased RR and longer PFS time, whereas OS did not show any clinical benefits when compared to standard chemotherapy [Articles:21783417, 20022809, 20573926]. Similarly, in a phase III trial where previously untreated East Asian NSCLC patients who were nonsmoker/former light smokers and treated with gefinitib or carboplatin/paclitaxel (IPASS trial), activating mutation harboring patients treated with gefitinib had significantly longer PFS time compared to carboplatin/paclitaxel group; on the contrary, EGFR mutation negative group had significantly shorter PFS time when treated with gefinitib [Articles:19692680, 21670455]. OS did not differ between two treatments arm (gefitinb vs. carboplatin/paclitaxel)[Article:21670455].
Few studies compared the clinical benefits of common L858R (rs121434568) and exon 19 mutations and failed to show any differential benefits [Articles:21670455, 17106442, 17610986, 16785471] between two types of mutations, except one small study suggested exon 19 deletions group had longer PFS compared to L858R in TKIs treated NSCLC patients [Article:21725039].
Demographic differences of the incidence of EGFR mutations in NSCLC patients were observed. Activating mutations in EGFR are more frequent in women (38% vs. 10% in man), nonsmokers (47% vs. 7% in smokers), adenocarcinomas (30% vs. 2% in non-adenocarcinoma) and Asian populations (26-36% vs. 7-12% in Whites) [Articles:20952405, 19147750, 17888036, 16850125]. EGFR mutations in all NSCLC patients (whether smokers or not) will be important, as inhibition of this receptor has considerable clinical benefits [Article:16850125]. This observation was particularly clear in Asian patients with EGFR mutations treated with gefitinib in the IPASS trial [Article:19692680].
T790M (rs121434569): Acquired resistance to TKIs: The majority of patients with an activating EGFR mutation received clinical benefits when treated with erlotinib/gefitinib, but the magnitude and the duration of the clinical response significantly vary among NSCLC patients [Articles:22594511, 23022519]. Mutation type (L858R vs. exon 19 del) seems to have little effect on the clinical outcome [Articles:21670455, 17106442, 17610986, 16785471]. However, majority of the NSCLC patients will develop resistance to erlotinib/gefitinib treatment and progress, this situation greatly limits the ability of these drugs to significantly prolong patient survival [Articles:22594511, 23022519].
The most frequent mechanism of acquired resistance to TKIs is the T790M (rs121434569) mutation [Articles:15737014, 15728811, 21430269, 18093943, 16258541, 17020982, 17085664, 18981003, 18992959, 19381876, 19589612, 20129249, 21248300, 21921847]. This mutation may reduce the binding capability of TKIs to the TK domain of EGFR by an allosteric mechanism [Article:15728811] and increase the affinity to ATP that requires much higher concentration of TKIs to inhibit EGFR [Article:18227510]. The T790M mutation was originally thought to be acquired by tumors cells during treatment with TKIs, however, when more sensitive methods were used for mutation detection, the presence of T790M mutation was shown in a small fraction of tumors cells before treatment with TKIs and usually co-exists in these cells with other activating mutations [Articles:21248300, 16912157]. The tumor cell clones carrying both the activating and the T790M mutations will eventually develop resistance to the TKIs and will be responsible for the progression or recurrence of the disease, this hypothesis was confirmed in which patients harboring T790M mutation before the start of the treatment had a significantly shorter PFS [Articles:22215752, 21233402, 18596266] and decreased response rate (RR) [Article:16912157] compared with those not having T790M mutation.
The T790M (rs121434569) mutation, along with other secondary mutations in EGFR, was observed as a germline mutation in four siblings of European descent family in which multiple members developed NSCLC [Article:16258541]. Neither T790M mutation was observed in a cohort of ~400 subjects [Article:16258541], nor dbSNP (build 137) presented its existence in general population.
Other resistance mechanism to EGFR-targeted therapy:
The T790M mutation is detectable in about 50% of patients with NSCLC patients who develop resistance to TKIs treatment [Articles:21430269, 18093943, 21248300], and may not explain all resistance cases. One of the mechanisms of resistance to TKIs involves the MET gene amplification that occurs 5-20% of patients [Article:17463250]. MET gene amplification leads to EGFR-independent activation of the PI3K/AKT pathway through MET/ErbB-3 heterodimers and may be responsible for the resistance to TKIs [Article:17463250]. MET amplification in NSCLC was identified in a very small proportion of tumor cells even before exposure to TKIs and this population of cells expands following TKIs treatment [Article:20129249]. The other mechanism involves the KRAS oncogene, which is mutated in approximately 15-30% of NSCLC [Article:16043828]. Mutations in KRAS and those in EGFR seem to be mutually exclusive, and KRAS mutation harboring patients do not respond to TKI therapy [Article:18804418]. KRAS is a downstream mediator of EGFR-induced cell signaling, and mutations confer constitutive activation of the signaling pathway(s), independent of EGFR activation [Article:19636327]. Additional potential mechanisms of resistance to TKIs have been also identified [Article:23022519]. Thus, NSCLC has a significant level of plasticity, being able to activate several different mechanisms leading to resistance to EGFR-TKIs.
EGFR gene copy number and protein expression in NSCLC:
Mutations, gene copy number, and protein expression are three EGFR-related biomarkers that have been extensively studied in clinical trials in order to obtain better predictive and prognostic values for treatment modalities. Although EGFR gene amplification frequently correlates with EGFR protein overexpression and tumor progression [Article:18381415], EGFR gene amplification and protein overexpression studies yielded controversial results in terms of prognostic significance and clinical benefits [Articles:22594511, 23022519, 21969500]. In this respect, the IPASS study (>1200 NSCLC patients) is a cornerstone trial in the assessment of biomarkers associated with EGFR-TKIs activity [Article:21670455]. EGFR mutations are the strongest predictive biomarker for PFS and objective RR to first-line gefitinib versus carboplatin/paclitaxel treatment and post hoc analysis suggested that the predictive value of EGFR gene copy number was driven by coexisting EGFR mutation [Article:21670455].
EGFR contains over 800 SNPs found in >= 1% of samples (dbSNP build 137) and a few of them may have some biological importance.
Intron 1 (CA)n repeat (rs11568315): This is a simple sequence repeat polymorphism, dinucleotides range from 9 to 23, with majority clustered around 15 to 21 CA repeats (dbSNP build 137). Association of intron 1 CA repeat polymorphism to better clinical response in NSCLC patients treated with gefitinib was analyzed in four different studies, all has less than 100 study subjects [Articles:17375033, 19201048, 19473722, 17597605]. 16 or fewer CA repeats were considered short and combined together and, 17 or more CA repeats were considered long. NSCLC patients carrying one or two short alleles are more likely to have better clinical response (increased RR, increased PFS and increased OS) when treated with gefitinib as compared to patients who have two long alleles [Articles:17375033, 19201048, 19473722, 17597605]. Well-powered studies are needed to replicate the beneficial clinical effect of rs11568315 in NSCLC patients.
The -216G>T (rs712829): Patients with GT+TT genotypes are associated with increased PFS time when treated with gefitinib in NSCLC patients [Article:17375033] and, decreased severity of diarrhea when treated with erlotinib in neoplasm patients [Article:18309947] as compared patients with GG genotypes. Both studies involved few patients and well-powered studies are needed to replicate suggested associations [Articles:17375033, 18309947].
GWAS studies on tumor risk and EGFR: Two well-powered genome wide association studies (GWAS) showed that SNPs in EGFR were significantly associated to risk of glioma, most common primary brain tumors [Articles:21531791, 22886559], implications of these finding on treatment of glioma or other cancers are yet to be seen.
Extensive molecular, cancer genome sequencing and recent GWAS studies showed that EGFR is an important gene for many biological process and tumorigenesis. Better clinical output can be obtained in NSCLC patients who are harboring activating somatic EGFR mutations who are treated with TKIs. Nevertheless, additional therapies are needed for those patients who are wild type for the EGFR gene. Clinical & treatment associations with germline EGFR SNPs are not strong, more studies are necessary to clarify the role of germline SNPs in treatment of NSCLC.