All these trials reported a significant improvement in terms of PFS, but no statistical difference was seen for OS, maybe because of the high percentage of crossover from standard therapy to the experimental one after disease progression.
All these trials reported a significant improvement in terms of PFS, but no statistical difference was seen for OS, maybe because of the high percentage of crossover from standard therapy to the experimental one after disease progression.
2. Mechanisms of Resistance
Although all generations of EGFR-TKIs have been proven to be very effective for NSCLC with common EGFR mutations, almost 5–25% of these patients do not experience a clinical benefit with these drugs due to intrinsic resistance
[46][47]. On the other hand, the major part of patients treated with EGFR-TKIs became resistant to these therapies despite an initial response or stable disease. The various mechanisms of resistance to EGFR-TKIs could be explained by the high molecular heterogeneity of NSCLCs (
Figure 1 and
Figure 2)
[48]. Therefore, deepening the knowledge about the EGFR-TKI resistance mechanisms is one of the most-important aims in order to improve the treatment strategy of these patients.
Figure 1. Mutations of resistance to EGFR-TKIs, according to the generation of TKIs and the line of therapy.
Figure 2. EGFR signaling pathway and EGFR-TKIs’ resistance mechanism.
2.1. Intrinsic Resistance
Patients with intrinsic resistance report an early tumour progression without prior tumour response; some of them respond for a very short period (<3 months)
[49].
A possible cause regards the pharmacokinetics. In detail, treatment can fail due to the ineffective drug dose in the target area. This event can occur because of drug competition or the difficulty of first-/second-generation EGFR-TKIs to reach sanctuary localisations, such as the brain
[50]. However, intrinsic resistance is often due to the lack of a target dependency (i.e., EGFR exon 20 mutations) or the genes alterations from other pathways (downstream or parallel pathways)
[51][52]. Mechanisms of intrinsic resistance have been reported above all in patients with uncommon mutations and, more rarely, with common ones
[51][52]. This type of resistance often depends on the presence of a drug-resistant EGFR mutation; the most-important ones are the exon 20 insertions (1–10% of all EGFR mutations) and the T790M EGFR mutation (approximately <1–65% of cases, based on the detection method employed)
[53][54][55][56]. The lack of first-/second-generation EGFR-TKIs’ efficacy for the T790M mutation and exon 20 insertions is the reason for the development of third-generation EGFR-TKIs and EGFR-Ins20-specific inhibitors, respectively.
2.1.1. Intrinsic Resistance to First-/Second-Generation EGFR-TKIs
Exon 20 insertions correspond to the addition of residues at the N-lobe of EGFR (M766 to C775), while the C-helix (A767 to C775) is their preferential location
[57]. This area is fundamental to regulate ATP and EGFR-TKI binding with the consequent activation of the kinase domain through a conformation change
[57]. Commonly, exon 20 insertion mutations led to a reduced sensitivity to EGFR-TKIs; however, in vitro studies described that some insertion mutations, such as the insertion EGFR-A763_Y764insFQEA, confer high sensitivity to EGFR-TKIs
[58][59]. These data have been confirmed by clinical trials in which NSCLC patients with some types of insertion mutations experienced prolonged periods of disease control under EGFR-TKI treatment
[58].
The presence of the EGFR T790M mutation at diagnosis is a rare event, which suggests, in some cases, a germinal EGFR mutation
[55][56][60]. It is associated with the worst response to first- or second-generation EGFR-TKIs and poor clinical outcomes
[55][56][61]. The EGFR T790M mutation regards exon 20 and consists of a substitution of the threonine at position 790 with a methionine in the ATP-binding pocket of the kinase domain. This change prevents the binding of EGFR-TKIs to the receptor due to a steric hindrance; on the other hand, it leads to an in increased affinity between ATP and EGFR. Therefore, the receptor affinity for ATP becomes greater than that for the drug with a severe reduction in EGFR-TKI activity
[62].
Another EGFR mutation that is responsible for intrinsic resistance to EGFR-TKIs in vitro is the variant III (vIII) in-frame deletion of exons 2–7 in the extracellular domain
[63]. This mutation is present in almost 5% of human lung squamous cell carcinoma and determines the unsuccessful binding of EGF and other growth factors to EGFRvIII
[64][65]. The reason behind the constitutive activation of EGFRvIII and the EGFR-TKI resistance is probably the structural changes of the EGFR protein affecting the ATP pocket and the intracellular domain conformation
[63].
Some genetic alterations could occur in NSCLC patients with common EGFR mutations with the consequent reduction of sensitivity to EGFR-TKI therapy. In this regard, BIM is a proapoptotic member of the Bcl-2 family that plays a critical role in apoptosis mediated by EGFR-TKIs
[66]. NSCLC patients with deletion polymorphisms or low-to-intermediate levels of BIM mRNA have poor clinical efficacy under EGFR-TKIs
[66][67]. Furthermore, low levels of NF1 and the overexpression of RhoB are correlated with poor clinical efficacy
[68][69]. Moreover, the plasma detection of TP53 gene co-mutations within two months of EGFR-TKI treatments is related to the worst PFS and OS
[70]. CRIPTO1 is a member of the EGF-CFC family; it is a cell membrane protein linked to glycosylphosphatidylinositol. High basal levels of CRIPTO1 lead to a reduced EGFR-TKI sensitivity through the activation of ZEB1 and SRC. In this way, ZEB1 promotes epithelial-to-mesenchymal transition (EMT), while SCR stimulates AKT and MEK signalling
[71].
The major part of oncogenic driver mutations in NSCLC is mutually exclusive, although some of them are present simultaneously, such as PI3KCA or TP53 mutations, with some other oncogenic driver mutations
[72][73]. If, on the one hand, co-mutations of the PI3KCA and EGFR genes have no clinical impact
[72], on the other hand, the co-mutation of EGFR Del19 and non-disruptive TP53 exon 8 is correlated with intrinsic resistance to first-generation EGFR-TKIs
[73].
In some cases, pretreatment AXL and CDCP1 RNA overexpression coexist with EGFR mutations and correlate with poor response to first-generation EGFR-TKIs
[74] and, likewise, co-alterations in some cell cycle genes or genes of the PI3K, MAPK, and Wnt/β-catenin pathways
[16][49].
2.1.2. Intrinsic Resistance to Third-Generation EGFR-TKIs
Although most studies regard resistance to osimertinib during second-line therapy, some literature data report intrinsic resistance when it is administered as a first-line treatment
[75]. In this regard, the transformation of NSCLC to SCLC has been considered a possible mechanism of intrinsic resistance
[76].
The HER2 and MET genes’ amplification was associated in in vitro studies with reduced sensitivity to third-generation EGFR-TKIs such as osimertinib and rolecitinib
[77][78]. The combination of the KRAS G12D mutation and PTEN loss was also detected in NSCLC patients with intrinsic resistance to osimertinib
[49]. The worst response to third-generation EGFR-TKIs was reported also in NSCLC patients with the EGFR mutation and CDCP1 or AXL RNA overexpression at baseline
[74].
2.2. Acquired Resistance
All the patients treated with EGFR-TKIs experience a progression of disease (PD) after a variable period of treatment. Patients usually develop acquired resistance after 9–12 months of treatment with first-/second-generation EGFR-TKIs, almost 10 months with second-line third-generation EGFR-TKIs
[42], and about 19 months with first-line third-generation EGFR-TKIs
[27].
Clinical criteria of acquired resistance to EGFR-TKIs in NSCLC patients have been proposed by Jackman et al., although further clinical validation is needed. The criteria by Gandara et al. are based on the type of PD: central nervous system (CNS), systemic, and oligo-progression. In the consideration of the undefined management of NSCLC patients who progressed to EGFR-TKIs because of acquired resistance, this classification could help clinicians establish the best treatment strategy based on the PD patterns
[77]. For example, the same treatment with EGFR-TKI could be continued in patients with slow-PD and without clinical deterioration. A similar strategy could be applied for those patients with CNS PD or oligo-PD in association with local treatment to the site of progression (e.g., radiotherapy or surgery)
[79].
The comprehension of the mechanisms leading to the acquired resistance is complex due to different aspects such as: (1) the type of EGFR-TKI; (2) the line of treatment with a specific EGFR-TKI; (3) the tumour biology, in particular histology, intrinsic mutability, microenvironment, and the type of initial EGFR mutation.
In the literature, there are several studies regarding the development of acquired resistance to the first-line treatment with first- or second-generation EGFR-TKIs or second-line treatment with third-generation EGFR-TKIs, usually due to the occurrence of the T790M mutation. In contrast, few data have been published about the acquired resistance to first-line osimertinib treatment. However, given the increasingly larger number of patients who will be treated with this drug, it is crucial to deepen the knowledge about the biological mechanisms of EGFR-TKI resistance.
Below,
wresearche
rs describe the acquired resistance mechanisms to EGFR-TKIs known today. In detail, they can be classified into EGFR-dependent due to the insurgence of new EGFR mutations and EGFR-independent mechanisms due to the activation of alternative pathways.
2.2.1. EGFR-Dependent Mechanisms: Secondary EGFR Mutations
Acquired resistance based on EGFR-dependent mechanisms is due to the insurgence of secondary and tertiary mutations and/or amplifications of the EGFR gene with the consequent alteration of the receptor aminoacidic structure. Therefore, this leads to a conformational change that can regard the kinase or the ATP-binding pocket of the mutant EGFR, limiting drug accessibility or increasing the ATP affinity.
The incidence of EGFR-dependent acquired resistance is variable based on the type of EGFR-TKI administered and the line of treatment. To be specific, approximately 50% of patients develop this type of resistance under first-/second-generation EGFR-TKIs, 20% of them if they are treated with third-generation EGFR-TKI as a second-line therapy, and 10–15% with first-line third-generation EGFR-TKIs
[80].
EGFR T790 Mutation
This is the most-frequent (49–63%) secondary mutation resulting in the insurgence of acquired resistance under treatment with first-/second-generation EGFR-TKIs
[81]. Therefore, third-generation inhibitors were specifically designed to target the EGFR T790 mutation.
The EGFR T790M mutation regards exon 20 and consists of a substitution of the threonine at position 790 with a methionine in the ATP-binding pocket of the kinase domain. This change prevents the binding of EGFR-TKIs to the receptor due to a steric hindrance; on the other hand, it leads to an increased affinity between ATP and EGFR. Therefore, the receptor affinity for ATP becomes greater than that for the drug with a severe reduction in EGFR-TKI activity
[82].
Some studies have hypothesised that this type of resistance might depend on the selection of pre-existing drug-resistant EGFR-T790M-positive clones during treatment with first-/second-generation EGFR-TKIs or on de novo acquisition of the EGFR-T790M mutation by initially drug-tolerant cells, negative for the EGFR T790M mutation
[83]. Experimental data on gefitinib-resistant PC9 cells showed that the early EGFR T790M mutant clones derived from pre-existing EGFR T790M mutated cells were selected for gefitinib treatment. The other theory includes the late de novo occurrence of this type of mutation in drug-tolerant cells due to the prolonged exposure to a first-/second-generation EGFR inhibitor
[53]. In an in vitro study, Hata et al. documented the restorations of late-emerging T790M cells’ sensitivity to third-generation EGFR-TKIs thanks to the treatment of tumour cells with navitoclax, an inhibitor of the antiapoptotic factors BCL-2 and BCL-xL
[84].
Approximately 43% of NSCLC patients lose the EGFR T790M mutation with the PD
[30][53][57]. This event suggests the existence of subclones with the EGFR T790M mutation
[77]. Usually, the loss of T790M is associated with the presence of exon 19 deletion (83%) and, only rarely, with the L858R mutation (14%)
[57]. Moreover, from a clinical point of view, this event at the time of progression is correlated with the worst clinical outcomes
[23][85][86][87]. From a molecular point of view, it is associated with the loss of EGFR dependence and dependence on non-EGFR mechanisms
[85].
Tertiary EGFR Mutations: Resistance to Second-Line Third-Generation EGFR-TKI
The AURA3 trial was the first study that showed the insurgence of acquired resistance to second-line osimertinib by means of the employment of cell-free DNA (cfDNA) genomic profiles
[85][88]. To be specific, the results demonstrated that about 50% of the NSCLC patients maintained the EGFR T790M mutation, including those that experienced the insurgence of the tertiary EGFR mutation. The authors reported that acquired tertiary EGFR mutations occurred in 21% of cases, and the most-common one (15%) was the EGFR exon 20 C797S mutation
[42][89][90]. In contrast, the FLAURA study, in which osimertinib was administered as a first-line therapy, reported a C797S mutation frequency of 7%.
The C797S mutation corresponds to a substitution in the ATP-binding site of a cysteine with a serine at codon 797, resulting in the inability of osimertinib to covalently bond with the mutant EGFR
[91]. Moreover, some studies showed that this mutation also prevents the binding of other irreversible third-generation EGFR-TKIs such as olmutinib, rociletinib, and narzatinib to the EGFR active site
[92][93]. Interestingly, the allelic context of the C797S mutation can predict the response to subsequent EGFR-TKI therapies. In detail, when NSCLC patients have the T790M and C797S mutations on the same allele (
cis-mutations), they experience resistance to all available generations of EGFR-TKIs as a single agent or combined with other drugs
[93][94]. On the other hand, when patients have these mutations on different alleles (
trans-mutations), they experience sensitivity to first- and second-generation EGFR-TKIs. However, mutations in trans are rare, regarding less than 30% of cases
[94][95].
Rare point EGFR mutations in exon 20 have been also identified in the C796 residue such as the G796R (0.56% of patients with lung adenocarcinoma treated with osimertinib), G796S, and G796D mutations, which are adjacent to C797 in exon 20 and can sterically impair the binding of osimertinib to EGFR
[92][96][97][98].
L792 exon 20 mutations, including L792H, L792Y, and L792F, consist of the addition of a benzene or imidazole ring to the side chain of L792, resulting in the binding disruption of osimertinib to the EGFR kinase domain
[97][99]. This mutation usually is located in cis with T790M, but less frequently, it can also occur in trans with EGFR C796/C797X mutations
[94].
The L718Q, L718V, and L798I mutations in exon 18 affect the ATP-binding site of the EGFR kinase domain. Therefore, they determine steric restriction, preventing the binding of osimertinib
[86][94][99]. These mutations are responsible for osimertinib resistance independent of the C797 mutation; in fact, they are not co-existent. L718Q/V are associated with sensitivity to first- and second-generation EGFR-TKIs, above all when T790M has been lost
[100]. Osimertinib resistance is also caused by the G719A mutation located close to the L718 residue
[94].
G724S is a very rare EGFR mutation located in exon 20, usually associated with EGFR exon 19 deletion. This mutation regards the P-loop of the kinase domain interfering with the binding of osimertinib
[101][102][103]. However, this altered structure does not confer resistance to second-generation EGFR inhibitors
[51].
SV768IL (S768I + V769L) is another rare (3%) mutation of EGFR exon 20 that has been identified in second-line therapy with osimertinib
[90].
Rarely, tertiary EGFR mutations such as G724S, L718Q, V834L, and L718V can occur in patients that lost the T790M mutation
[86].
Secondary EGFR Mutations: Resistance to First-Line Third-Generation EGFR-TKI
FLAURA was the first study that evaluated resistance to osimertinib as a first-line treatment
[27][104]. Other literature data derive from some case reports or small case series
[85][105].
This phase 3 trial analysed cfDNA samples through NGS, but no emergent T790M mutation has been detected. This discovery is in line with the well-known activity of osimertinib towards EGFR-sensitising and T790M mutations
[104].
In this study, EGFR mutation/amplification was rare (9%), as well as C797S mutation frequency (7%), although it is the most-common mechanism after MET amplification (15%)
[90]. S768I or combined EGFR mutation, such as Del19 + G724S (exon18), L718Q + EGFR ex20ins (exon 18 + 20), C797X, or S768I (exon 20), or L718Q + C797S, L718Q + L797S (exon 18 + 20) are very rare secondary mutations, each corresponding to about 1% of cases
[11][12][104][106].
Interestingly, this
res
tudyearch gave evidence that the mechanisms of resistance to first-line osimertinib depend on EGFR only for a small proportion of cases, and no EGFR T790M mutation was observed. Alternatively, EGFR-dependent mechanisms of acquired resistance are typical for those patients who receive osimertinib as a second-line therapy. Therefore, the T790M mutation will be less common due to the more-frequent use of osimertinib as the first-line therapy.
Rare EGFR Mutations
Although the underlying mechanisms are not well-defined yet, literature data described other rare EGFR point mutations that are responsible for acquired resistance to first-/second-generation EGFR-TKIs and regard less than 10% of NSCLC patients. They include D761Y and L747S (exon 19) or T854A (exon 21), Asp761Tyr, 39 Thr854Ala, and 40 Leu747Ser
[60][99][107].
Other rare molecular alterations, such as the β-catenin mutation, have been detected in association with the EGFR T790M mutation
[81].