A secondary T790M mutation is the most important mechanism of acquired drug resistance against first-generation EGFR-TKIs. The crystal structure of the ATP binding pocket is altered due to this mutation, inhibiting the binding of TKIs and ATP. Thus, downstream signal transduction cannot be inhibited by TKIs, and these drugs do not subsequently restrict tumor growth (20,21). The mechanism of action of the first-generation EGFR-TKI differs from that of the second-generation EGFR-TKI; the second-generation EGFR-TKI irreversibly binds to the ErbB receptor, resulting in a more potent effect than the first-generation drugs (22), but the mechanism of drug resistance is similar (23–25). In the ARCHER1050 study, dacomitinib had overall survival (OS) benefits relative to gefitinib in the Chinese population (median overall survival (mOS) duration was 32.5 months vs. 24.9 months, P=0.0097) (26). The LUX-Lung7 trial compared the efficacy of afatinib and gefitinib for the treatment of NSCLC patients with EGFR mutations. The results showed that the progression-free survival (PFS) duration of the afatinib group was longer than that of the gefitinib group (11.0 vs. 10.9 months; P=0.017) (11). These results suggest that compared with first-generation EGFR-TKIs, the effects of second-generation EGFR-TKIs are longer in the context of T790M.

Osimertinib is the most widely used third-generation EGFR-TKI and it can effectively and selectively inhibit tumors with EGFR-sensitive and T790M drug-resistant mutations (27), exhibiting a significant effect in NSCLC patients with brain metastases (28,29). Almonertinib (30,31) and furmonertinib (32,33) have also been approved in China, and several other third-generation EGFR-TKI inhibitors are in different stages of research and development (Table I). Lazertinib achieved a 57% overall response rate (ORR) in the T790M (+) population in a phase 2 clinical trial (34). The drug exhibited a potent beneficial effect on brain lesions, and the intracranial disease control rate in the entire population was 90.6% (35). In January 2021, the Korean Food and Drug Administration (MFDS) approved the listing of lazertinib for the treatment of patients with locally advanced or metastatic NSCLC positive for EGFR T790M mutations who previously received EGFR-TKI treatment (36). The third-generation EGFR-TKIs olmutinib (37–39) and nazartinib (40) are also approved in South Korea.

Other rare secondary mutations, such as L747S (41), D761Y (42), and T854A (43), have also been reported to be associated with gefitinib or erlotinib resistance. Due to the low incidence of these mutations, there are few in vitro studies and case reports showing whether Osimertinib is effective against these rare mutations (44–47).

Activation of HER2, also known as ERBB2, triggers functional abnormalities in several downstream signaling pathways, such as the mitogen-activated protein kinase (MAPK), inosine phosphate 3-kinase (PI3K)/protein kinase B (AKT), protein kinase C (PKC), and signal transducer and transcriptional activator (STAT) pathways, resulting in uncontrolled cell proliferation (48,49). HER2 overexpression occurs in ~12% of NSCLC patients who are resistant to first- and second-generation EGFR-TKIs and usually do not co-exist with the T790M secondary mutation (50). Standard treatment for managing this drug resistance mechanism is currently not available, and there is insufficient evidence to show that existing anti-HER2 therapies are effective. The selective HER2 tyrosine kinase inhibitors poziotinib (51) and pyrotinib (52,53), and the antibody conjugate drugs T-DM1 (54) and trastuzumabe-deruxtecan (55) are potential treatment options.

MET is a proto-oncogene and one of the key driver genes in several types of cancer (56). The MET gene encodes c-Met [a hepatocyte growth factor (HGF) receptor], which is responsible for regulating important processes, such as cell differentiation, proliferation, migration, and apoptosis (57). Hepatocyte growth factor (HGF) binds to c-Met to phosphorylate tyrosine kinase residues in the catalytic domain; activates the downstream pathways modulated by PI3K, MAPK, and STAT3 signaling, and promotes cell transformation, cell invasion, cell proliferation, and cell cycle progression (57,58). MET amplification accounts for 2–4% of untreated NSCLC cases (59) and for 5–20% of patients with acquired drug resistance against first- and second-generation EGFR-TKIs (58,60,61). Lai et al (62) showed that an increased copy number of the MET gene is not equal to MET amplification; only MET amplification is a determinant of EGFR-TKI resistance in NSCLC patients.

Due to the crosstalk between MET and RTK (EGFR) signaling pathways (63), it has been proposed that the combination of MET-TKIs and EGFR-TKIs may be a solution for MET-driven EGFR-TKI resistance (64). After disease progression in the context of EGFR-TKI treatment, patients with MET amplification were treated with camatinib combined with gefitinib. The ORR was 29%, and the PFS was 5.5 months (65). Tepotinib combined with gefitinib prolonged the survival time compared with chemotherapy (mOS: 37.3 vs. 13.1 months) (66). Sequist et al (67) evaluated the efficacy of Osimertinib + savolitinib in two global expansion cohorts (part B and part D) of the Tatton study, and the results showed a higher response rate in subgroup B3 (previously untreated with third-generation EGFR-TKIs and positive for T790M) and subgroup D (previously untreated with third-generation EGFR-TKIs), with ORR values of 67 and 64% and median (m)PFS values of 11 and 9.1 months, respectively. Osimertinib combined with savolitinib is also a potential treatment approach (67). In addition to the above combination of treatments, telisotuzumab-vedotin combined with erlotinib (68), capmatinib combined with erlotinib (69), and emibetuzumab combined with erlotinib (70) also achieved certain benefits.

PIK3A can induce the phosphorylation and subsequent activation of the downstream AKT signal transduction pathway and it plays a central role in regulating tumor cell growth, reproduction, migration, and apoptosis (71). The role of PIK3CA mutations in NSCLC remains contested. Some researchers consider PIK3CA mutations to be an independent risk factor for NSCLC patient survival (72), and the survival time of patients with EGFR and PIK3CA mutations treated with EGFR-TKIs was shown to be shorter than that of people with only EGFR mutations (73). However, it has also been shown that PIK3CA mutations have no significant effect on NSCLC patient survival times (74). PIK3CA mutations are a mechanism of acquired EGFR-TKI resistance in patients with EGFR-mutated lung cancer (75). The frequency of PIK3CA mutations after EGFR-TKI resistance is 2–3% (76). Preclinical studies have found that double targeting of MEK and PI3K can effectively control the proliferation of EGFR-TKI drug-resistant NSCLC cell lines (77). Alpelisib (a PI3K inhibitor) has been approved by the Food and Drug Administration (FDA) for the treatment of breast cancer (78), but it has not been applied for NSCLC after the development of resistance to TKIs.

KRAS mutations activate downstream pathways, such as the MAPK and PI3K pathways, driving the occurrence and development of tumors (79). The proportion of KRAS mutations after the development of EGFR-TKI resistance is ~1% (76). Tanaka et al (80) suggested that the mechanism underlying KRASG12C-acquired drug resistance to KRAS-TKI is related to the activation of the RAS-MAPK signals and the production of KRASY96D resistance genes. The FDA approved the KRASG12C inhibitor sotorasib in May 2021 to treat NSCLC patients with KRASG12C mutations after at least one previous systematic treatment (81).

BRAF mutations increase the activity of RAF kinase, activates downstream MEK, and regulates cell growth, proliferation, differentiation, migration, and apoptosis (82). BRAFV600E is the most common BRAF mutation, accounting for 36% of all BRAF mutations (83). BRAF mutations account for only 1% of patients with acquired drug resistance to TKIs (84). Dabrafenib combined with trametinib has been approved by the FDA for the treatment of metastatic NSCLC with BRAFV600E mutations (85).

The AXL-mediated Gas6/Axl signaling pathway is associated with tumor cell growth, metastasis, invasion, EMT, angiogenesis, drug resistance, immune regulation, and stem cell maintenance (86,87). In 2012, a study found that Axl expression was upregulated in patients with acquired drug resistance to EGFR-TKIs, and EGFR-TKI sensitivity was restored after blocking Axl (88). Thus, Axl is a promising therapeutic target for patients with acquired drug resistance. Small molecule inhibitors, monoclonal antibodies, and antibody-drug conjugates targeting Axl are currently under development (89). DS-1205 (an AXL inhibitor) combined with gefitinib (90) and BGB324 (an AXL inhibitor) combined with erlotinib (91) were evaluated, and preliminary results were promising.

PTEN negatively regulates the PTEN/PI3K/Akt signaling pathway and regulates cell growth, apoptosis, and migration (92). Studies have shown that patients with EGFR mutations with PTEN deletions have significantly shorter PFS durations than those without PTEN deletions (6 vs. 18 months) (93). According to Xun et al (92), the deletion of PTEN in lung cancer promotes the carcinogenic function of STMN1 (overexpression of which is related to tumor growth, metastasis, and poor survival) through the PI3K/AKT pathway. Other reported drug resistance mechanisms include loss of neurofibromin 1 activity (94), amplification of the CT10 homologous oncogene of v-crk avian sarcoma virus (95), a multistep mechanism involved in the insulin-like growth factor 1 receptor (IGF1R) pathway (96), and the fibroblast growth factor (FGF) 2/FGF receptor 1 (FGFR1) autocrine growth pathway (97). As these drug resistance mutations are rare, no drugs targeting them have been approved.

Compound mutations indicate the presence of more than one EGFR mutation, either common or uncommon, within the same tumor. Attili et al (98) found high heterogeneity in the incidence of compound mutations (4–26% of total EGFR mutant cases), with the variance possibly due to the different testing methods adopted, and the specific mutations considered. In various combinations, compound EGFR mutations containing either exon 21 p. L858R or exon 19 deletions were common (99). The response rate of those tumors with compound mutations to EGFR-TKIs compared with those with single mutations is contested. Rossi et al (100) found a longer mOS in the compound mutation group than in the single rare mutation group (33.6 vs. 12 months; P=0.473), whereas Jiang et al (101) concluded that patients in the single mutation group exhibited a longer mOS than those in the co-mutation group (ORR: 64.6% vs. 27.4%, P<0.001). More prospective randomized clinical trials (RCTs) are required to reconcile these differences.

A co-mutation is defined as the coexistence of an EGFR mutation along with one or more other gene mutations. The co-mutation incidence rate was 66.0% in the retrospective study of Jiang (101). Co-mutations, including TP53 (102,103), HER family genes (104), KRAS, MET, and ROS1, are typically considered to be associated with poor prognosis (105,106).

The Aura3 study revealed that 49% of the patients had T790M loss, and 14% had EGFRC797S mutations, the most common mutations acquired after the development of Osimertinib resistance (154). EGFRC797S mutations include cis (98%) and trans mutations (2%). T790M and C797S mutations that occur simultaneously in the same allele are referred to as cis mutations, and mutations that occur in different alleles are referred to as trans mutations (157,158).

Recent studies have shown that a third-generation TKI combined with a first-generation EGFR-TKI can change the expression profile of drug resistance genes in lung adenocarcinoma patients with EGFR activation mutations, and T790M and trans-C797S triple mutations (158). Brigatinib combined with cetuximab is an effective treatment strategy for these lung adenocarcinoma patients with EGFR activation mutations and T790M and cis-C797S triple mutations (158,159). Chang et al (160) reported the case of a patient with lung adenocarcinoma with triple mutations (L858R, T790M, and cis-G796s/cis-C797s). After treatment failure with brigatinib combined with cetuximab, the patient responded to the combination of brigatinib, Osimertinib, and bevacizumab. Other reported treatments include Osimertinib combined with anlotinib (161), chemotherapy combined with antiangiogenic agents (162), and apatinib combined with afatinib (163). At present, fourth-generation EGFR-TKIs targeting drug-resistant T790M mutations are under development, although no drug has been approved. Fourth-generation EGFR-TKIs in clinical trials and currently undergoing research and development and are described in subsequent sections.

In a study where next-generation sequencing (NGS) analysis was performed on 93 samples obtained after the development of Osimertinib resistance, EGFRG796/C797, L792, and L718/G719 mutations were found in 24.7, 10.8, and 9.7% of cases, respectively (164). G724 mutations were also reported in some studies (165,166). At present, a drug that targets the aforementioned mutated genes is not available. In vitro studies have confirmed that L792 is still sensitive to gefitinib (167) and that tumors with the L718Q mutations remain sensitive to icotinib (168). In a patient with the EGFRG724S/19del mutation after second-line Osimertinib resistance, PFS was achieved after using afatinib for 3.8 months (169).

EGFR-independent mechanisms of drug resistance primarily include activation of abnormal accessory and downstream pathways and histological/phenotypic transformation. Most of the mechanisms of Osimertinib drug resistance are the same as those of first- and second-generation TKIs.

Leonetti et al (155) demonstrated that the incidence of MET amplification after Osimertinib first-line treatment resistance was 7–15%, and that of MET amplification after second-line treatment resistance was 5–50%. In the B1 expansion cohort of the TATTON study (previously treated with third-generation EGFR-TKIs), a 5.5-month PFS duration was obtained with Osimertinib combined with sevotinib (67), and other therapeutic developments have been described. Zhang et al (170) found that MET amplification weakened the response of lung tumors to immunotherapy by inhibiting the STING signaling pathway, and that MET inhibitors combined with immune checkpoint inhibitors overcame this drug resistance; however, this information needs to be confirmed by further prospective studies. Amivantamab (JNJ-61186372) is a bispecific antibody against EGFR and MET that has been approved for the treatment of patients with EGFR exon 20 insertion mutations (171). Amivantamab can inhibit both the phosphorylation of EGFR and MET and the activation of downstream signals and has potent antibody-dependent cell-mediated cytotoxic effects (171,172). Amivantamab is inhibited by double targeting, which showed an inhibitory effect on several types of mutations secondary to EGFR-TKI resistance (C797S mutations, MET amplification, previous resistance to Osimertinib) (173).

HER2 amplification is one of the mechanisms of Osimertinib resistance (174). The AURA3 study showed that HER2 amplification was detected in 5% (4 out of 73) of second-line Osimertinib-resistant patients (157). In the FLAURA study, HER2 amplification occurred in 2% of first-line Osimertinib-resistant patients (175). PIK3CA mutations occur in patients with Osimertinib resistance, and T790M mutations may be retained or lost. Moreover, PIK3CA mutations showed different incidences in different studies (176,177).

Abnormal FGFR expression can lead to the activation of the FGFR cancer-related signaling pathway effectors (PI3K/AKT, STAT, and MAPK) and affect cell proliferation, survival, metabolism, and migration as well as the cell cycle (178). In vitro studies found that hypoxia can lead to acquired resistance to EGFR-TKIs by increasing the expression of FGFR1 (179). The combined use of EGFR-TKIs and FGFR1 inhibitors (BGJ398) may represent a potential therapeutic strategy for the management of NSCLC (179,180). Upregulation of IGF1R is one of the mechanisms of drug resistance to EGFR-TKIs, including Osimertinib (181–183). In cells resistant to Osimertinib that exhibit low levels of AXL expression, short-term IGF-1R inhibition combined with Osimertinib can eradicate tumors and prevent regrowth (184).

Histological transformation from lung adenocarcinoma to SCLC, SCC and EMT was also observed in patients with Osimertinib resistance (177,185). Platinum-containing dual-drug chemotherapy is still recommended for these patients.