Understanding the treatment response and resistance to targeted therapies in non-small cell lung cancer: clinical insights and perspectives

Lung cancer remains the leading cause of mortality worldwide. Non-small cell lung cancer (NSCLC) is the most common subtype of lung cancer with a generally poor prognosis. In recent years, advances in targeted therapy and sequencing technology have brought significant improvement in the therapeutic outcomes of patients with advanced NSCLC. Targeted inhibitors directed against specific mutated or rearranged oncogenes, such as epidermal growth factor receptor (EGFR), anaplastic lymphoma kinase (ALK), and receptor tyrosine kinase ROS proto-oncogene 1(ROS1) among others, exhibit promising anti-tumor activity. Unfortunately, some patients develop acquired resistance and disease progression soon after initial remission. Despite the continuous development of new drugs and strategies to overcome drug resistance, it is still a major challenge in the treatment of NSCLC. The landscape of targeted therapy for NSCLC is evolving rapidly in response to the pace of scientific research. This study aimed to provide a comprehensive review of tumor target antigens and agents related to targeted therapy in NSCLC.


Introduction
In recent years, targeted therapy has achieved significant success in advanced non-small cell lung cancer (NSCLC).Patients with metastatic lung cancer who qualify for targeted therapies now experience prolonged survival, with 5-year survival rates ranging from 15% to 60%, contingent on the specific biomarker identified (1)(2)(3)(4).Consequently, molecular and immune biomarker testing of lung cancer specimens is crucial to identifying potentially effective targeted treatments, especially in patients with metastatic NSCLC (3)(4)(5)(6)(7).It aims to alleviate symptoms, decrease tumor burden, and improve overall survival (OS).
Here we summarize the essential therapeutic targets and targeted drugs for NSCLC (Table 1) and provide insights into the treatment response and resistance mechanisms associated with targeted therapies.
2 Biomarkers and target therapies 2.1 EGFR inhibitors EGFR is the most common driver gene in NSCLC.The mutation frequency is approximately 10-15% in Western Europe and North America and can be as high as 30%-50% in East Asia (9,10).Common EGFR mutations involve exon 19 deletions and the exon 21 mutation p.L858R, while less frequent mutations include p.S768I/V, p.L861X, and p.G719X (11) (Figure 1).

First-generation medications
Gefitinib and erlotinib were both reversible inhibitors of the firstgeneration EGFR TKIs.They can selectively and reversibly prevent ATP binding, thereby inhibiting EGFR autophosphorylation (12).An analysis of five clinical studies in which erlotinib or gefitinib was used as first-line treatment in NSCLC (stage IIIB or IV) revealed that the response rate was 67% in patients with sensitizing EGFR mutations (13).

Erlotinib
Erlotinib has shown better efficacy than conventional chemotherapy in advanced NSCLC patients with EGFR mutations in multiple randomized phase III trials.In the EURTAC trial, patients receiving erlotinib demonstrated a response rate of 58% with a median PFS of 9.7 months, whereas those receiving conventional chemotherapy exhibited a response rate of 15% with a median PFS of 5.2 months (14).In the trial CALGB30406,

Gefitinib
The phase III randomized trial IPASS evaluated the efficacy of gefitinib in previously untreated NSCLC patients in East Asia, patients treated with gefitinib exhibited a significantly high objective response rate of 71.2% compared to those treated with carboplatin-paclitaxel (17).The OPTIMAL trial also reported a superior response rate in the gefitinib group compared to the chemotherapy group (83% vs. 36%) (18).The phase III randomized trial WJOG5108L reported similar response rates for gefitinib and erlotinib at 55.0% and 58.9%, respectively (19).

Afatinib
Afatinib, a second-generation oral TKI, exerts irreversible inhibition targeting the ErbB/HER receptor family including EGFR and HER2 (20).In a phase IIB trial comparing afatinib and gefitinib for first-line treatment in common EGFR mutation metastatic adenocarcinoma patients, afatinib demonstrated a significantly higher objective tumor response rate compared to gefitinib (70% vs. 56%) (21).Updated results revealed no significant difference in OS between the two groups (22).A subgroup analysis of several LUX-LUNG trials (LUX-LUNG 2, 3, and 6) evaluated the efficacy of afatinib in patients with mutationpositive metastatic NSCLC.The response rate was 77.8% in patients with EGFR p.G719X mutation, 100% in p.S768I, and 56.3% in p.L861Q (23).Notably, these findings should be interpreted cautiously as treatment crossover occurred in most patients (72% in LUX-LUNG 3 and 80% in LUX-LUNG 6).

Dacomitinib
Dacomitinib is a second-generation oral TKI, that exerts irreversible inhibition on ErbB/HER receptors, including EGFR, HER1, HER2, and HER4.In the phase III randomized trial ARCHER1050, patients receiving dacomitinib as first-line treatment exhibited an objective response rate of 75% (24).Subsequent updated data indicated that dacomitinib-treated patients experienced longer progression-free survival (PFS) (14.7 months vs. 9.2 months) and OS (34.1 months vs. 27 months) compared to the gefitinib group (25,26).

Third-generation medications
Approximately 60% of patients who progressed on firstand second-generation EGFR TKI treatment harbor EGFR p.T790M mutation.The third-generation EGFR TKIs were originally designed to overcome the resistance caused by acquired EGFR p.T790M mutation.Osimertinib, an oral and irreversible TKI, exhibits selectivity for both common EGFR mutations and p.T790M mutation, with activity within the central nervous system (CNS) (27)(28)(29).Osimertinib is the first third-generation EGFR-TKI approved by the Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for metastatic NSCLC patients with EGFR p.T790M mutation (30).
In a phase III randomized trial (AURA3), involving patients with EGFR p.T790M-positive metastatic NSCLC progressing after first-line treatment, the objective response rate was significantly superior with osimertinib (71%) compared to chemotherapy (31%).Osimertinib also exhibited a longer PFS (10.1 vs. 4.4 months).Notably, in the subgroup of patients with CNS metastases, osimertinib presented a prolonged PFS compared to those treated with platinum-pemetrexed (8.5 vs. 4.2 months) (31).The BLOOM study which increased the standard dose of osimertinib from 80 mg once daily to 160 mg once daily, have proved beneficial of the higher dose of osimertinib for patients with leptomeningeal disease The frequencies of EGFR mutations in NSCLC.
In addition to the second-or third-line use to overcome resistance of firstand second-generation EGFR TKI treatment, osimertinib has been used as first-line to treat EGFR mutant NSCLC patients.A Multicenter, Phase II Trial (KCSG-LU15-09) demonstrated an objective response rate of 50% for osimertinib as first-line treatment in 37 patients with EGFR rare mutations, including p.S768I, p.L861Q, and p.G719X (34).The phase III randomized trial (FLAURA) also proved a longer median OS with osimertinib as first-line treatment than with erlotinib or gefitinib (38.6 months vs. 31.8months), though the objective response rate was comparable (80% vs 76%) (35, 36).

Other medications 2.1.4.1 Amivantamab
Amivantamab is a bispecific human antibody to both EGFR and MET receptors that bypasses resistance to EGFR TKIs (37).CHRYSALIS study, a phase I study, evaluated the efficacy of Amivantamab-vmjw as a subsequent treatment in 81 metastatic NSCLC patients with EGFR exon 20 insertion.The overall response rate reported in this cohort was 40% (37).In a phase III study (PAPILLON), amivantamab-chemotherapy significantly improved PFS of patients with EGFR exon 20 insertions who had not received previous systemic therapy when compared to chemotherapy alone (median, 11.4 months and 6.7 months, respectively) (38).And MARIPOSA evaluated the therapeutic efficacy of Amivantamab plus carboplatin-pemetrexed (chemotherapy) with and without Lazertinib in patients with EGFR-mutated (exon 19 deletions or L858R) locally advanced or metastatic NSCLC after disease progression on Osimertinib.The median PFS was significantly longer for amivantamab-chemotherapy and amivantamab-lazertinibchemotherapy versus chemotherapy (6.3 and 8.3 versus 4.2 months, respectively) (39).

Mobocertinib
Mobocertinib is an oral TKI selectively inhibiting EGFR and HER2 exon 20 insertion mutations (40,41).A phase I/II study evaluated the efficacy of mobocertinib as a subsequent treatment in patients with EGFR exon 20 insertion mutation.The objective response rate was 28%, with a median duration of response of 17.5 months and a median PFS of 7.3 months (40).Subsequently, mobocertinib received FDA accelerated approval for advanced or metastatic NSCLC in adults with EGFR exon 20 insertion mutations who progressed during or after platinum-based chemotherapy.
However, results from the phase III trial, EXCLAIM-2, indicated that the objective response rates and disease control rates between the mobocertinib and chemotherapy groups are similar (response rate: 32% vs. 30%, control rate: 87% vs. 80%) (42).As a result, the FDA and Takeda withdrew mobocertinib in America in October 2023, as it did not meet the primary endpoint of the study.

Cetuximab
Cetuximab is a monoclonal antibody to EGFR.In a large phase III randomized trial, FLEX, the combination of chemotherapy and cetuximab proved higher overall response rates than chemotherapy alone (36% vs. 29%) and comparable median OS (11.3 vs. 10.1 months) (43).However, this combination exhibited poorer tolerability considering the nearly 40% incidence of grade 4 neutropenia.Therefore, the use of cetuximab is not yet recommended in NSCLC.

First-generation medications 2.2.1.1 Crizotinib
Crizotinib is a first-generation oral TKI and the first TKI approved for treating ALK-positive NSCLC, effectively inhibits ALK rearrangements, ROS1 rearrangements, high-level MET amplification, and METex14 skipping mutations.In phase I and II studies, crizotinib demonstrated objective tumor responses in approximately 60% of ALK-positive NSCLC patients, with a median PFS ranging from 7 to 10 months (46)(47)(48).A phase III randomized study, PROFILE 1014, assessing the efficacy of crizotinib as first-line targeted therapy, yielded promising results with an objective response rate of 74% (49).For ALKpositive patients progressing after first-line chemotherapy, crizotinib has shown efficacy in improving PFS (7.7 months) and enhancing response rates (65%) (50).
The ALEX trial, a phase III randomized study, compared the efficacy of alectinib and crizotinib as first-line treatments in 303 ALK-positive advanced NSCLC patients, including those with asymptomatic brain metastases.The response rate in the alectinib group was 82.9% and 75.5% in the crizotinib group (53).Another phase III trial, J-ALEX, enrolled 207 ALK inhibitor-naive Japanese patients with ALK-positive NSCLC, also proved that alectinib as a first-line treatment achieved a higher objective response rate compared to crizotinib (92% vs. 79%) (54).
Efficacy of alectinib as subsequent treatments was reported by phase II trials with a total response rate of 48% to 50% in metastatic NSCLC patients with ALK rearrangement progressing after crizotinib treatment (55,56).

Brigatinib
Brigatinib is a second-generation TKI that inhibits a broad spectrum of ALK rearrangements.As first-line treatment, brigatinib was reported a higher systemic objective response rate of 71% than crizotinib (60%) in the ALTA-1L trial.The intracranial response rate was also notably higher with brigatinib (78%) compared to crizotinib (29%) (57).Updated data further confirmed that the 3year PFS in the brigatinib group was superior to crizotinib (43% vs. 19%) (58).
A phase II study, ALTA, evaluated the efficacy of two different doses of brigatinib in ALK-positive metastatic NSCLC patients who had experienced disease progression on or intolerance to crizotinib.The overall response rate ranged from 45% to 54%.In patients with measurable brain metastases, the intracranial overall response rate was observed to be between 42% and 67% (59, 60).

Ceritinib
Ceritinib is a second-generation oral TKI designed for ALK and ROS1 rearrangements (61), showing promising results in various clinical trials.In the ASCEND-4 trial, the overall response to ceritinib as first-line therapy was 72•5% with a median PFS of 16.6 months, as compared with 26•7% with a median PFS of 8.1 months in the chemotherapy group (62).
As subsequent treatment in patients with prior exposure to at least two treatments, ceritinib was reported an overall response rate of 38.6%, with a concurrent intracranial response rate of 45.0% in a phase II study (ASCEND-2) (63), and a higher overall response rate of 45% than pemetrexed or docetaxel chemotherapy (8%) (64).

Third-generation medications
Lorlatinib, a third-generation oral TKI with excellent CNS penetration, selectively inhibits ALK and ROS1.It exhibits the ability to inhibit ALK resistance mutations that emerge following treatment with first and second-generation ALK inhibitors (65-68).In the phase III randomized trial, CROWN, lorlatinib demonstrated complete CNS responses in 61% of patients with baseline brain metastases, compared to only 15% with crizotinib (69).Updated data reveals a lower cumulative CNS progression rate with lorlatinib (7%) than crizotinib (72%) over 12 months, and higher 1-year PFS rates of 78% than 22% for crizotinib in patients with brain metastases (70).
Lorlatinib also remains effective for patients experiencing progression after treatment of other ALK inhibitors, especially those with CNS involvement.Among patients with measurable baseline CNS lesions, 47% achieved objective responses, and 63% achieved an objective intracranial response (66, 67).

Dabrafenib/trametinib
In a phase II trial, dabrafenib/trametinib as first-line treatment demonstrated a robust overall response rate of 64% in 36 patients with BRAF p.V600E mutation (76).An updated analysis of this trial revealed a 5-year OS rate of 22% (1).Another dual-cohort phase II study conducted a comparative analysis between patients receiving dabrafenib monotherapy and combination therapy with dabrafenib and trametinib.The results indicated a distinct overall response rate of 33% and 67%, and median PFS durations of 5.5 months and 10.2 months, respectively (77).

Encorafenib/binimetinib
In the PHAROS trial, an open-label, multicenter, single-arm study, an impressive overall response rate of 75% was observed among the 59 treatment-naive patients with BRAF p.V600E mutation, with a median duration of response not achieved.In the cohort of 39 previously treated patients, the overall response rate was 46%, and the median duration of response was 16.7 months (78).

ERBB2 (HER2) inhibitors 2.4.1 Ado-trastuzumab emtansine
Ado-Trastuzumab Emtansine, also known as T-DM1, is a humanized antibody-drug conjugate comprising the HER2targeting antibody trastuzumab and the microtubule inhibitor emtansine (79).In a phase II basket trial, the efficacy of adotrastuzumab emtansine was assessed in patients with metastatic NSCLC and HER2 mutations, revealing a partial response rate of 44% (79, 80).Another study focused on patients with HER2 exon20 insertion mutations, indicating an objective response rate of 38% with ado-trastuzumab emtansine (81).These findings underscore the potential of ado-trastuzumab emtansine as a targeted therapeutic option for patients with HER2-mutated NSCLC.

KRAS inhibitors
KRAS mutations are identified in approximately 30% of NSCLC patients (84).These mutations are predominantly (>95%) located at codons 12 and 13.The p.G12C variant was the most prevalent, constituting 39% of all KRAS mutations, followed by p.G12V (21%) and p.G12D (17%) variants (85).Sotorasib and adagrasib are both an oral inhibitor to the RAS GTPase family, demonstrating efficacy in inhibiting the KRAS p.G12C mutation in patients with metastatic NSCLC who have previously undergone chemotherapy (± immunotherapy).

Sotorasib
Sotorasib, as a small-molecule inhibitor, irreversibly binds to the non-active GDP pocket of KRAS, forming an irreversible covalent bond with the cysteine residue in KRAS p.G12C.This covalent interaction locks the protein in an inactive state.By disrupting the KRAS signaling pathway, sotorasib inhibits cell growth as well as tumor progression both in vitro and in vivo and induces apoptosis in KRAS p.G12C tumor cell lines (86,87).
In a phase II study involving 126 patients with KRAS p.G12Cpositive advanced NSCLC who had prior platinum-based chemotherapy (with or without immunotherapy), subsequent treatment with sotorasib showed a partial response rate of 33.9% and complete response rate of 4.2% (88).The phase III randomized study, CodeBreaK200 trial, has also reported the efficacy of sotorasib in patients in a similar situation (89).Sotorasib demonstrated a significantly higher overall response rate of 28.1% than docetaxel (13.2%).Moreover, the disease-control rate in the sotorasib group was 82.5%, compared to 60.3% in the docetaxel group.

Adagrasib
In a phase II study involving 116 patients who had previously undergone platinum-based chemotherapy with or without immunotherapy, adagrasib demonstrated an objective response rate of 42.9%.The efficacy of adagrasib in cases with KRAS mutations beyond p.G12C remains to be systematically evaluated (90).

MET inhibitors
The oncogenic driver genomic alterations associated with MET comprise METex14 skipping mutations and high-level MET amplification.High-level MET amplification was recently identified as an emerging biomarker.Its definition may vary depending on the reagent kits.When employing Next-Generation Sequencing (NGS), high-level MET amplification is defined as the copy number greater than 10 (91).The FDA has not yet approved recommended drugs for NSCLC patients carrying these mutations, despite their approval in other tumor types.

Capmatinib
Capmatinib is an oral TKI selectively targeting MET alterations.The GEOMETRY trial revealed that capmatinib achieved an overall response rate of 68% as a first-line treatment, and 41% as subsequent treatment in patients with METex14 skipping mutations.While in patients with high-level MET amplification, the response rate was 40% as the first-line therapy, and 29% as subsequent therapy (91).Notably, the updated data of GEOMETRY indicate that capmatinib exhibits anti-tumor efficacy within the brain (92).Another study revealed an overall response rate of 50% in a cohort of 10 patients with high-level MET amplification (93).

Crizotinib
Crizotinib is an oral TKI that inhibits METex14 skipping mutation and high-level MET amplification.A phase II study evaluated the efficacy of crizotinib in 69 patients with METex14 skipping mutations.The objective response rate was 32%, with a median PFS of 7.3 months (94).The PROFILE1001 study investigated the efficacy of crizotinib in advanced NSCLC patients with varying levels of MET amplification.Patients with MET genomic copy number over 10 demonstrated an overall response rate of 29% (95).

Tepotinib
Tepotinib is a selective oral TKI that inhibits METex14 skipping mutation and high-level MET amplification.A phase II study (VISION) assessed the efficacy of tepotinib in patients with MET mutations.The response rate in patients with METex14 skipping mutations was 46%.Another cohort comprising 24 patients with MET amplification but lacking METex14 skipping mutations exhibited an overall response rate of 41.7% (96, 97).

Larotrectinib
A study comprising 55 patients with various solid tumors and positive NTRK gene fusions revealed an overall response rate of 75% with larotrectinib (98).The updated data demonstrated that 90% of patients still remained alive one year after treatment.Furthermore, among 35 NTRK fusion cancer patients, the overall response rate reached 74% (102).

RET inhibitors
The RET gene is observed in 1-2% of all NSCLC patients with chromosomal rearrangements and is involved in various fusion partners such as KIF5B, TRIM33, CCDC6, and NCOA4 (103, 104).

Pralsetinib
In a phase I/II study (ARROW), pralsetinib was assessed in metastatic NSCLC patients with RET rearrangements.The overall response rate of pralsetinib was 70% as a first-line treatment, and 61% as a subsequent treatment reached 61% (105).The FDA approved pralsetinib in 2020 for the treatment of metastatic RET fusion-positive NSCLC patients.It is the first oral TKI targeting RET fusions (106).

Selpercatinib
A phase I/II study, Libretto-001, along with its updated results, reveals that selpercatinib exhibits remarkable efficacy in NSCLC patients with RET rearrangements.The overall response rate for first-line treatment was 85%, while 64% for subsequent treatment.Notably, in patients with brain metastases, selpercatinib demonstrated effectiveness in 91% of cases (107,108).

Cabozantinib
In a prospective phase II trial involving 26 RET fusion-positive patients treated with cabozantinib, the overall response rate was 28% (109, 110).

ROS1 inhibitors 2.9.1 Crizotinib
Crizotinib is a multitargeted inhibitor targeting MET, ALK, and ROS1.In an early-phase study, crizotinib demonstrated considerable efficacy in ROS1-rearranged NSCLC (111).The objective response rate in the expansion cohort treated with crizotinib reached 72%.The overall response duration was 17.6 months, with a median PFS of 19.2 months (112).
Three phase II studies confirmed an overall response rate of more than 70% with crizotinib in patients with ROS1 rearrangement.A phase II trial evaluating the efficacy of crizotinib in 127 East Asian patients reported an overall response rate of 72% (113).The PROFILE 1001 study and updated data reported an objective response rate of 72% in 53 ROS1-positive advanced NSCLC patients, including 3 complete responses and 33 partial responses (4,114).The multicenter trial, EUCROSS study, reported a total response rate of 70% in 30 patients treated with crizotinib (115).Additionally, a retrospective study assessing crizotinib in stage IV ROS1-rearranged NSCLC patients (n=30) reported an overall response rate of 80%, with a median PFS of 9.1 months (116).

Lorlatinib
Lorlatinib is an oral third-generation TKI targeting both ALK and ROS1 with significant CNS penetration.It was evaluated in a phase I/II trial for its efficacy in ROS1-positive metastatic NSCLC patients.The objective response rate in patients previously treated with crizotinib reached 35%, while treatment-naive patients demonstrated a 62% objective response rate.Notably, intracranial responses were observed in 50% of patients with prior crizotinib treatment and 64% of treatment-naive patients (65).

Entrectinib
Entrectinib is an oral TKI inhibiting multiple tyrosine kinases, including ROS1 and TRK.A pooled analysis of 53 patients with ROS1 rearrangement across several phase I and II trials (STARTRK-2 trial, STARTRK-1 trial, ALKA-372-001 trial) who received entrectinib as first-line treatment demonstrated an overall response rate of 77%, with a 55% intracranial response rate (100,101,117).Although entrectinib exhibits superior CNS penetration compared to crizotinib, it comes with higher toxicity, with an incidence of grade 3 or 4 adverse events of 34% (117).

Ceritinib
Ceritinib is a second-generation oral TKI inhibiting ALK and ROS1 rearrangements.In a phase II trial assessing ceritinib as firstline treatment in ROS1-rearranged NSCLC patients (28 evaluable patients), the reported overall response rate was 62%, with 1 case of complete response and 19 cases of partial responses (61).

Repotrectinib
A phase I/II trial assessed the efficacy and safety of repotrectinib in patients with advanced ROS1 fusion-positive NSCLC.The confirmed overall response rate was 79% among ROS1 TKI-naive patients and 38% among patients previously treated with other ROS1 inhibitors.Notably, responses were observed in intracranial lesions in patients with measurable CNS metastases, as well as in those with resistance mutations following TKI therapy (118).

VEGF or VEGF receptors inhibitors 2.10.1 Bevacizumab
Bevacizumab is a recombinant monoclonal antibody targeting VEGF.In a phase III randomized trial, ECOG4599, involving relapsed or advanced non-squamous NSCLC, the corresponding response rates were 35% in patients treated with a combination of bevacizumab chemotherapy and 15% in those treated with chemotherapy alone (119).Another phase III trial, NEJ026, compared the efficacy of erlotinib combined with bevacizumab to erlotinib monotherapy as first-line treatments in EGFR-positive advanced non-squamous NSCLC patients.The objective response rates were similar (erlotinib/Ramucirumab: 72% vs. erlotinib monotherapy: 67%) (120).

Ramucirumab
Ramucirumab is a recombinant monoclonal antibody targeting VEGF receptors.In the phase III randomized trial RELAY, first-line treatment with erlotinib/ramucirumab was compared to erlotinib monotherapy in EGFR-mutated advanced NSCLC patients.The overall response rates were similar (erlotinib/ramucirumab: 76% vs. erlotinib monotherapy: 75%) (121).The REVEL trial, a phase III randomized study in metastatic NSCLC patients who experienced disease progression, evaluated the efficacy of ramucirumab/docetaxel compared to docetaxel alone as subsequent therapy.The ramucirumab/docetaxel group exhibited higher overall response rates (23% vs. 14%) and disease control rates (64% vs. 53%) (122).

Nintedanib
Nintedanib is a potent, oral angiokinase inhibitor that targets the pro-angiogenic pathways mediated by VEGFR1-3 (123).In the phase III randomized controlled trial LUME-Lung 1, 1314 stage IIIB/IV patients progressing after first-line chemotherapy were randomly assigned to receive docetaxel plus nintedanib (n=655) or docetaxel plus placebo therapy (n=659).PFS was significantly improved in the nintedanib plus docetaxel group when compared to the docetaxel plus placebo group (median 3.4 months vs. 2.7 months) (PMID: (124)).
3 Resistance to targeted therapy

Overview of the mechanisms of resistance to targeted therapies
Resistance to targeted therapies is categorized as either primary (intrinsic) or secondary (acquired) (125).Primary resistance describes a de novo lack of therapeutic response, while secondary resistance indicates disease progression after the initial response.Despite distinct resistance mechanisms identified in patients with different gene alterations, there are common mechanisms shared among these cohorts (126).The acquired resistance mechanisms can be broadly classified into two categories.
The first category involves the development of additional genetic alterations in the primary oncogenes, activating continued downstream signaling.This is often attributed to secondary mutations in kinase targets or gene amplifications of the kinase itself (127).The second category of resistance development can occur independently of changes in the target gene.This scenario includes upregulation of bypass signaling pathways, histological changes of tumor tissue, or alterations in drug metabolism (128,129).Moreover, about 14% of small-cell lung cancer can histologically transform into NSCLC, often accompanied by resistance to the original TKI (130,131).
In 2010, Jackman et al. proposed the criteria of acquired resistance in EGFR-mutant NSCLC (132): 1) Patients must have previously received EGFR inhibitor treatment.2) Patients harbor either tumor-genotyping confirmed typical EGFR mutations associated with drug sensitivity, or objective clinical benefit from treatment with an EGFR inhibitor.3) Patients develop systemic progression while on continuous treatment with gefitinib or erlotinib within the last 30 days.4) No additional systemic treatment between cessation of EGFR inhibitor and initiation of new therapy.

Primary resistance
Primary resistance to EGFR inhibitors may be partially attributed to differential TKI sensitivity for different EGFR mutations.Typical EGFR mutations, including exon 19 deletions and p.L858R, are associated with significant sensitivity to TKIs (128).Conversely, exon 20 insertions or duplications, accounting for about 4% of patients with EGFR mutations, appear to have resistance to EGFR inhibitors (133).
Threonine 790 serves as the "gatekeeper" residue, crucial for inhibitor specificity in the ATP binding pocket.The p.T790M mutation activates wild-type (WT) EGFR, introducing an increase in the ATP affinity of the p.L858R mutant by more than an order of magnitude.This is the main mechanism by which the p.T790M mutation confers TKI resistance, reducing the efficacy of any ATPcompetitive kinase inhibitor.Irreversible inhibitors can simply overcome this resistance through covalent binding rather than alternative binding (141).Therefore, in patients with EGFR p.T790M-positive metastatic NSCLC experiencing progression after first-line treatment, osimertinib as an irreversible EGFR-TKI can achieve an objective response rate of over 70% (31).
Other secondary mutations include p.D761Y, p.L747S, and p.T854A.They reduce the sensitivity to EGFR inhibitors, but the resistance mechanism remains unknown (142).In the AURA trial, the acquired p.C797S mutation was observed in 14% of the samples (31).The p.C797S mutation frequency was 7% when osimertinib was used as first-line therapy (35).The EGFR p.C797S mutation, in which cysteine at codon 797 is replaced by serine in the ATP-binding site, results in the loss of the covalent bond between osimertinib and mutated EGFR.Predictably, the p.C797S mutation also leads to crossresistance by preventing other irreversible third-generation TKIs from binding to the EGFR active site (143)(144)(145).

Resistance to ALK inhibitors
The primary resistance to ALK inhibitors may be due to the different sensitivity of EML4::ALK variants and other ALK fusion genes to ALK inhibitors (148).Acquired resistance to ALK inhibitors typically occurs within the first year of treatment (125).Secondary mutations in the enzyme are the common mechanism of TKI resistance.It is noteworthy that multiple secondary mutations can occur in ALK-positive patients upon TKI resistance.The first "gatekeeper" mutation identified in the EML4::ALK kinase domain is p.L1196M (149).The substitution of leucine for methionine at position 1196 in the ATP binding pocket generates a mutated large amino acid side chain, which hinders crizotinib from binding to its receptor.Other identified acquired resistance point mutations include p.G1128A, p.1151Tins, p.L1152P/R, p.C1156Y, p.I1171T/ N/S, p.F1174V, p.V1180L, p.G1202R, p.S1206Y/C, p.E1210K, and p.G1269A (150)(151)(152)(153)(154)(155).
Numerous studies suggest that second-generation drugs such as alectinib, ceritinib, brigatinib, and ensatinib may be more effective than chemotherapy when treating NSCLC patients with no response to first-generation ALK inhibitors (64,(156)(157)(158).In patients treated with second-generation ALK inhibitors, the p.G1202R mutation is the most common secondary ALK mutation, appearing in 21% of ceritinib-treated patients, 29% of alectinib-treated patients, and 43% of brigatinib-treated patients (159).
A gain in ALK gene fusion copy number (more than two-fold increase) has recently been proposed as a mechanism of resistance to crizotinib in both in vitro and in patients (150,155).Based on single circulating tumor cell sequencing, another study reported repeated mutations in the RTK-KRAS (EGFR, KRAS, BRAF genes), TP53, and other genes in the ALK-independent pathway in crizotinib-resistant patients (160).
Resistance to ALK inhibitors can also occur through the activation of bypass signaling pathways, including YAP transcription co-regulator, EGFR signaling, KIT amplification, the IGF-1R pathway, MAPK amplification, the BRAF p.V600E mutation, and MET amplification (155, 161-165).MET amplification was observed in 15% of tumor samples from patients progressing after second-generation ALK inhibitors, and in 12% and 22% of tumor biopsy samples from patients progressing on second-generation inhibitors or lorlatinib, respectively (166).
The p.D2033N mutation causes the substitution of aspartate for asparagine at position 2033 in the ROS1 kinase hinge region, thus leading to significant resistance to ROS1 inhibitors in vitro (173,174).The p.L2026M and p.G2032R mutations in the ROS1 kinase domain confer crizotinib resistance by altering the "gatekeeper" position of ROS1 inhibitor binding (168, 175).Additionally, p.S1986F/Y in the kinase domain disrupts crucial activation sites, thereby increasing kinase activity.p.L2155S is anticipated to confer crizotinib resistance through protein dysfunction (176).
The mutations and/or copy number increases of genes in other RTKs or downstream MAPK pathway are also involved in the mechanism of resistance to ROS1 inhibitor (177).Mediators involved in this pathway include KRAS, NRAS, EGFR, HER2, MET, KIT, BRAF, and MEK, either as downstream or bypass mediators (167,168,172,174).KRAS p.G12D and BRAF p.V600E mutations are associated with crizotinib treatment, while NRAS p.Q61K is associated with entrectinib treatment (178).

Strategies for overcoming resistance to TKIs
Targeted therapies have significantly improved the prognosis of NSCLC patients with relevant genetic alterations, which is a major progress in the history of NSCLC treatment.However, part of the patients acquires TKI resistance and disease progression shortly after initial remission.Strategies have been investigated to overcoming resistance to TKIs, which include the continuation of TKI therapy beyond disease progression, combination with other TKIs, and the use of immune checkpoint inhibitors.

Continuation of TKI therapy beyond disease progression
A phase II open-label single-arm trial named ASPIRATION reported that the post-progression erlotinib patients exhibited deeper responses, longer PFS, prolonged time from overall response to progression, and fewer new lung lesions (179).A retrospective analysis of 414 ALK-positive NSCLC patients enrolled in PROFILE 1001 and PROFILE 1005 showed that continuation of crizotinib (>3 weeks) after progression conferred extended progression time and longer OS (180).However, more evidence supports the timely detection of potential resistance mutations and prompt switching to sensitive targeted therapies after disease progression.

Combination with other TKIs
In a phase Ib/II single-arm trial, 47% of EGFR TKI-resistant NSCLC patients with MET gene amplification and 32% of EGFR TKI-resistant patients with MET overexpression responded to the MET inhibitor capmatinib in combination with EGFR TKI (181).In another phase 1b study of the combination of the MET inhibitors savolitinib and gefitinib, up to 52% of patients with EGFR TKIresistant NSCLC with MET gene amplification had an objective response to the combination treatment regimen (182).In the subsequent INSIGHT study, 67% of EGFR TKI-resistant NSCLC patients with MET gene amplification had an objective therapeutic response to treatment with the MET inhibitor tepotinib combined with gefitinib (183).And in the phase Ib trial of the TATTON study, 64% of NSCLC patients who were resistant to firstor secondgeneration EGFR TKIs and had MET gene amplification showed improved response to savolitinib combined with osimertinib.However, only 30% of patients who were resistant to thirdgeneration EGFR TKIs and had MET gene amplification showed an objective response to this combination therapy (184).

Immune checkpoint inhibitors
In recent years, checkpoint inhibitor antibodies, including programmed cell death protein 1 (PD-1) inhibitors and programmed death ligand 1 (PD-L1) inhibitors, have demonstrated favorable outcomes in NSCLC treatment by blocking the PD-1 and PD-L1 interaction and enhancing the antitumor effects of endogenous T cells.Pembrolizumab, atezolizumab, and cemiplimab have all been reported to prolong PFS and OS in eligible patients (185)(186)(187)(188)(189).However, the efficacy of checkpoint inhibitor antibodies depends on the expression level of PD-L1, and for certain mutations such as EGFR exon 19 deletions, EGFR p.L858R mutations, or ALK rearrangements, they appeared to be less effective (190)(191)(192)(193)(194).
In conclusion, targeted therapy has brought significant benefits to NSCLC patients, but the emergence of TKI resistance poses a formidable obstacle.The treatment of NSCLC still has a long way to go.

TABLE 1
Summary of US FDA approved targeted therapies for non-small cell lung cancer.
erlotinib monotherapy achieved an impressive response rate of 70% (15).Another phase III trial reported a higher objective response rate in the gefitinib group compared to the chemotherapy group (73.7% vs. 30.7%)(16).