The EGFR or ErbB1 or human EGFR 1 (HER1) prevails as a member of receptor tyrosine kinases (RTKs) which are involved in the activation of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)-protein kinase B (AKT)-mammalian target of rapamycin (mTOR) signaling pathways, implicated in cell growth, proliferation and survival. Three major mechanisms in EGFR activation of these cell signaling pathways are aggravation of EGFR mutations in the malignant cells, enhanced expression of EGFRs, and finally either mutation of the specific target/ligand [epidermal growth factors (EGFs)] or their enhanced activities. The mutated receptor (EGFRs) and ligand (EGFs) fail to dissociate on releasing pro-survival and growth-promoting signals causing uncontrolled growth [19–21].

Statistically, the prevalence of EGFR oncogene mutations is 50% among Asian adenocarcinoma sufferers and 15% in the western patients. The two most common mutations of this gene are exon 19 deletion (60%) and L858R mis-sense mutations at position 858 (35%), wherein leucine is substituted by arginine, resulting in non-ligand bound EGFR activation. To date, erlotinib, gefitinib (1st generation), afatinib, dacomitinib (2nd generation), and osimertinib (3rd generation) are the FDA-approved drugs for EGFR inhibition (Table 1). Despite potent therapeutic programming, studies decipher a resistant manifestation for treatment with these TKIs, regulated via T790M secondary mutations, activation of erstwhile EGFR pathways and histological transformation [22, 23].

EGFR mutant NSCLC is characterized by a pathology wherein unlike the de novo T790M, the T790M prevails via exon 19 deletions than in L858R mutated EGFR-TKI resistant sufferers [24]. For selective TKI pressure, this mutated clone predominates. Perhaps, this eventuality led to the discovery of osimertinib, an EGFR-TKI that potently aggravates its sensitization as well as T790M resistant mutations. Studies have demonstrated improved outcomes for osimertinib therapy of T790M mutated NSCLCs besides enhanced PFS (18.9 months) and overall survival (OS, 38.6 months) in first-line settings [19, 20]. Recently, several studies reported osimertinib resistance, suggesting its resolving as a priority [21, 25, 26]. The EGFR mutated NSCLCs are normally treated with varying generation TKIs, depending on the specific mutation intensity. Several investigations have elucidated a greater efficacy of combinatorial treatment, comprising varied stoichiometries of EGFR-TKIs and CDs. A phase II study evaluated the osimertinib safety and efficacy via chemotherapy combination, for T790M mutation harboring NSCLC sufferers. Even though tolerable extents were improved, no progress in survival duration was noticed, making the investigators pursue a re-investigation [27]. Another phase III study evaluated osimertinib efficacy with and without platinum-pemetrexed chemotherapy, in EGFR-mutated advanced NSCLC sufferers. Except for minor AEs, the combination exhibited a manageable tolerance, making the analysis feasible for further studies [28].

Nearly 30% NSCLCs exhibit a mutated rat sarcoma (RAS) gene. RAS is an acronym of a gene having three isoforms, i.e., KRAS, neuroblastoma rasviral oncogene homolog (NRAS) and Harvey RAS viral oncogene homolog (HRAS). The product of this gene acts as a small guanosine triphosphate (GTP) binding protein by guanine exchange factors (GEFs), is involved in diverse functions including proliferation, survival and differentiation through their GTP hydrolases (GTPases) [43]. In the GTP-bound state, KRAS activates the downstream signaling pathways such as MAPKs/extracellular signal-regulated kinases (ERKs) and PI3K-AKT-mTOR pathway, aiding in cell survival and proliferation. Mutations of KRAS are more common in adenocarcinoma and can be identified using next-generation sequencing (NGS) [44]. Most KRAS mutations are manifested via codon 12 substitution (G12C), gathering attention for NSCLC treatment [45]. Contrary to a higher G12C prevalence in smokers, the KRAS G12D is more frequent in non-smokers [46]. Present KRAS mutations exhibiting NSCLC therapies rely on the non-oncogene driven NSCLC, comprising immunotherapy with or without platinum chemotherapy. Clinically approved drugs for KRAS G12C are sotorasib and adagrasib. In phase I/II trial of sotorasib (Codebreak 100), 59 KRAS, G12C mutated, metastatic NSCLC patients treated with a median of 3 previous therapies, were given sotorasib orally. Analysis revealed 32.2% outcome with a confirmed objective response (complete or partial) in 88.1% cases. The mPFS was 6.3 months with grade 3 or 4 treatment-related toxicity in 11.6% patients. Inspection revealed a significant response in pretreated advanced tumors exhibiting G12C mutations [47].

Presently, sotorasib is being tested via randomized phase III, Codebreak 200 trial comparative to docetaxel (NCT04303780), with PFS and OS as the primary and secondary endpoints [48]. Adagrasib is another KRAS G12C-specific TKI, for which a phase I/II (KRYSTAL-1) trial involving 79 pre-treated NSCLC patients, administered 600 mg adagrasib, twice daily has been completed [49]. Of the 51 sufferers listed for response, 45% ORR was noticed with grade 1–2 side effects. Several erstwhile KRAS G12C inhibitors are being evaluated in phase I/II clinical trials (JNJ-74699157 and Gadolinia-Doped Ceria-6036) [50, 51]. As less than 50% sufferers responded positively to sotorasib or adagrasib, it could be presumed that some patients exhibit intrinsic resistance to KRAS G12C inhibition. This assumption gathers support from the preclinical observations [52]. Another source of resistance to KRAS targeted NSCLC therapies could be their unequal distribution across the tumor sites in a single patient [53]. Studies also demonstrate the emergence of adaptive resistance due to KRAS TKIs selective pressure, exhibited via amplified upstream drivers, including RTKs/tyrosine kinaseproto-oncogene (Src), homology 2 domain-containing phosphatase 2 which manifests from KRAS inhibition. Experimental evidence for reduced ERK activity via KRAS G12C TKIs does establish a suppressed ERK-driven RTKs/Src homology phosphotyrosine phosphatase 2 (SHP2) inhibition, further regulating NRAS, HRAS, and KRAS G12C to activate the MAPK/ERK signaling [52, 54]. It could be a further certainty that tumor cells may no longer depend on RAS pathway for survival and proliferation [55]. Therefore, despite inadequate clinical data, there is a high probability of KRAS G12C inhibitors being ineffective in many sufferers. Intriguing curiosity prevails for screening KRAS G12C inhibitors in combination with ICIs [56, 57]. In several preclinical studies, these combinations are being pursued as KRAS G12C positive tumor cell lines exhibit an immunosuppressive environment attenuated by KRAS inhibition [58, 59]. Other combinations led to the preclinical screening of adagrasib with SHP2 inhibitor, TNO155 [60]. However, no conclusive results have yet been published. Targeting with others is being attempted, prominently, MRTX1133, a KRAS G12D inhibitor [61]. Another molecule being examined is BI 1701963, a KRAS activator inhibiting the KRAS pathway irrespective of underlying mutation [62]. Of the other major KRAS targeting mechanisms, a messenger RNA (mRNA) vaccine targeting KRAS mutant cells has already entered a phase I trial, emerging potent from the immune response in the preclinical studies [63, 64].

Rearrangements in anaplastic large cell lymphoma kinase (ALK) gene are witnessed in 2–7% advanced NSCLC sufferers, with a higher frequency in young non-smokers. The echinoderm microtubule-associated protein-like 4 (EML4) and ALK genes reside within the short arm of chromosome 2; inversion of which provided the novel fusion oncogene EML4-ALK [65, 66]. A mainstay in the transmembrane receptor kinases, ALK can activate multiple signaling cascades including PI3K-AKT, signaling adaptor protein crk-Guanine nucleotide exchange factor (Crk1-C3G), MAPK 2/3 MAPK 5 (MEK5)-ERK5, Janus kinase (JAK)-STAT pathways [67]. Major ALK alterations in NSCLC are characterized by point mutations, deletions and rearrangements leading to reactivation. The ALK rearrangements are manifested via interstitial deletion and inversion in chromosome 2p, resulting in EML4-ALK fusion gene product [68, 69]. Murine tumors and human cell lines expressing EML4-ALK have been screened towards ALK inhibitors [70, 71]. A striking observation herein has been the mutually exclusive (without EGFR and KRAS) prevalence of mutated ALK gene, illustrating a single gene product instigated malignant status [72, 73]. The frequent ALK rearrangements in LUADs are characterized by solid tumors and recurrent signet-ring cells rich in intracellular mucin, in the Western population [74]. Crizotinib is the first validated CD for targeted therapy of ALK-positive advanced NSCLC, enabling a 10.9-month PFS contrary to 7 months chemotherapy [75]. However, abysmal CNS penetration is the reason for crizotinib resistance, resolved using the 2nd generation ALK-TKIs (Table 2). The first amongst these is certinib, granted FDA approval in 2014 for ALK-positive NSCLC patients exhibiting crizotinib intolerance. First-line therapy approval for crizotinib was given in 2017 based on ASCEND-1 and ASCEND-2 studies [76, 77], with the latter accomplishing a 38.6% ORR to crizotinib pretreated ALK + NSCLC, via 750 mg daily intake [77]. A phase I, three-arm ASCEND-8 study showed that 450 mg certinib with food had a similar efficacy with much less gastrointestinal toxicity than 750 mg fasted, leading to its 450 mg OD approval with food [78]. However, subsequent randomized phase III trials, compared certinib with standard chemotherapy in first (ASCEND-4) and second-line (ASCEND-5) settings revealed multiple dose modifications due to AEs [79, 80].

Alectinib was the next screened ALK-TKIs, wherein ALEX trial inferred its preferential first-line treatment, ascertained via response of 303 untreated ALK-positive advanced NSCLC sufferers, randomized to receive alectinib or crizotinib. A 12-month analysis revealed a 68.4% survival for alectinib contrary to the 48.7% for crizotinib [81]. The observations were supported by 34.8-month mPFS for alectinib contrary to 10.9 months for crizotinib [82]. The mPFS with baseline CNS metastasis was 27.7 months with alectinib contrary to the 7.4 months 12-month PFS contrary to 43% for crizotinib. These observations were complemented by 71% ORR (60% for crizotinib) besides 78% intracranial response [83]. A further ALTA-1L study also supported the brigatinib efficacy, irrespective of ALK fusion variant or a tumor suppressor gene (TP53) mutation, in the sufferers with baseline brain metastasis [84]. Clinical efficacy of brigatinib (NCT02706626), is being analyzed for resistive ALK-mutations via treatment with other 2nd generation drugs [85]. A phase III trial (NCT03596866), evaluating brigatinib and alectinib efficacy in first-line ALK mutated NSCLC patients is currently ongoing [86]. The latest ALK-TKI being evaluated is ensartinib, a 2nd generation aminopyridine-containing small molecule reported for its MET, BAL, Axl, Ephrin type-A-receptor 2 (EPHA2), LTK, ROS1 and Ste20-like kinase (SLK) inhibitions [87]. A phase I/II study, comprising 97 advanced ALK-positive, NSCLC patients given 225 mg ensartinib daily, revealed rashness, nausea, vomiting, pruritus and fatigue as frequent aftermaths. The response rate and mPFS were 60%, 80% and 9.2 months, 26.2 months for ALK-TKIs evaluable and naive patients. Besides, sufferers exhibiting brain metastasis showed a 64% ORR [88]. A phase III clinical trial comparing ensartinib and crizotinib efficacy in ALK-positive NSCLC patients, neared its completion in 2018 [89].

A still potent, third-generation ALK and ROS1 inhibiting drug is lorlatinib, a small and compact macrocyclic inhibitor, restraining G1202R mutation but no compound aberrations [90]. A recent phase II trial observed a 47% ORR and a 63% objective intracranial response on 198 ALK-positive, advanced NSCLC sufferers with hypercholesterolemia, hypertriglyceridemia and peripheral neuropathy as prominent AEs [91]. A recent phase III CROWN analysis, comparing the lorlatinib and crizotinib efficacy showed a distinct PFS with 0.28 as hazard ratio, fetching lorlatinib the first-line FDA approval [92]. Current knowledge of lorlatinib is marred by inadequate comparative studies with 2nd generation drugs. Sequential use of “single mutant” active ALK TKIs, aggravates the risk of double ALK resistance, for which 4th generation ALK TKIs (TPX-0131, NVL-655) are being evaluated. TPX-0131 is a compact macrocyclic inhibitor that fits well in the ATP binding pocket and reduces the vulnerability to multiple ALK TKI-resistant mutations (solvent front, hinge region, gatekeeper, and compound mutations) [93]. NVL-655 is a brain penetrative small molecule inhibitor, confronting the solvent-dependent drug resistance (G1202R, G1202R-L1196M and G1201R + G1269A) [94]. Entrectinib and repotrectinib (TPX-0005) are the further potent ALK TKIs, specifically targeting tropomyosin receptor kinase (TRK) A/B/C, ALK, ROS1, and ALK, ROS1, TRK A/B/C [95, 96]. Results of phase I/II basket and STARTRK-1 trials reported adequate tolerance with selective potency for entrectinib [97]. Repotrectinib is a macrocyclic TKI, smaller than lorlatinib with a potent CNS activity. Its latest screening via TREDENT-1 study (NCT03093116) reveals an encouraging response in ALK mutant NSCLC [98].

ROS1 rearrangement is characterized by 1–2% NSCLC cases, noticed more in young never smokers. The locus for ROS1 resides on chromosome 6, encoding for a tyrosine kinase receptor with the rearrangement being exhibited by the sufferers with reasonably similar clinical symptoms to those of ALK-rearrangements. Crizotinib is the first distinct CD approved (March 2016) for ROS1 mutated advanced NSCLC. Several studies evaluating crizotinib efficacy are undergoing, wherein a phase I effort revealed significant antitumor efficacy. Screening divulged a 72% ORR with 17.6 months as median duration while with mPFS and OS of 19.3 months and 51.4 months [112]. No explicit claims could be made for crizotinib antitumor efficacy as more than 80% sufferers were earlier administered at least one previous line of treatments. On similar lines, phase II crizotinib trials in Europe and East Asia, exhibited a 63% ORR with significant reproducibility and safety [112, 113]. In an open-label, single arm trial of 127 East Asian patients with ROS1 rearranged NSCLC, 71.7% ORR was noted irrespective of brain metastasis or prior treatments [114]. A low response was correlated with a baseline CNS disease, with 18.2 months PFS compared to 10.2 months in the non-CNS baseline sufferers. Importantly, 18% sufferers exhibited a baseline brain metastasis, subject to neurological stability in the last 15 days.

ROS1 kinase abnormal NSCLC treatment has also been made with certinib, a second generation drug for the treatment of TKI-naive and crizotinib resistant ALK positive NSCLC [115]. Preclinical studies screened certinib as a selective ROS1 inhibitor with 20-fold potency over crizotinib [116–118]. A phase II clinical trial comprising 30 crizotinib naive, ROS1 positive NSCLC sufferers, demonstrated a 67% ORR with 21-month duration of response (DOR) and a 19-month mPFS [118]. Though there were 2 sufferers with a prior crizotinib therapy, no responses (NR) were observed and the trial was re-attempted on crizotinib-naive sufferers. A striking observation was the single crizotinib resistant and seven crizotinib-naive patients amongst the 8 sufferers having CNS vulnerability, with a 25% ORR and 63% disease control rate (DCR). These observations though projected certinib therapeutic utility but could not establish its efficacy in overcoming crizotinib resistance, with G2032R, D2033N, L1951R, and S1986Y/F as the troubling mutations [119–121]. Furthermore, the phase II trial on certinib revealed diarrhoea (78%), nausea (59%), anorexia (56%) and vomiting (53%) as prominent AEs to a 750 mg daily intake [111, 118, 122]. A subsequent ASCEND-8 study, however, elucidated certinib usefulness at 450 mg on daily basis, providing greater tolerance with unchanged clinical efficacy [123]. To counter the certinib inadequacy, ROS1 and ALK selective lorlatinib, was examined due to its significant CNS penetration besides the efficacy towards multiple ROS1 mutations [121, 124, 125]. In a lately single arm phase I/II trial screening 69 ROS1 positive NSCLC sufferers (30% ROS1 TKI naive, 58% earlier treated with crizotinib and 12% treated with one, non-crizotinib ROS1 TKI or more than 2 ROS1 TKIs), an overwhelming 57% of treated individuals exhibited brain metastases at baseline with 41% ORR amongst all. Thereby, lorlatinib efficacy was noted as substantially greater in TKI naive patients (ORR: 62%, PFS: 21 months) than crizotinib (ORR: 35%, PFS: 8.5 months). Although grade 3 or 4 AEs were witnessed in 49% sufferers, no deaths occurred.

To still counter the vulnerabilities, a multikinase inhibitor, entrectinib was proposed with significant potential towards ROS1, ALK, and TRK resistance [91, 126]. This drug exhibited a better blood-brain barrier trafficking and CNS prevalence [95]. The STARTRK-1, STARTRK-2, and ALKA-372-001 trials accorded the FDA approval to entrectinib via accelerated mode for metastatic ROS1 mutant NSCLC [127, 128]. Eventually, the findings were published in 2020 [129]. The trials screened 53, ROS1 inhibitor-naive patients for ORR and DOR as primary endpoints while PFS, OS, intracranial ORR and DOR were recorded as secondary endpoints. Majority of the sufferers were females (64%), with 59% never-smokers exhibiting an ECOG (functioning ability involving self-care, daily activity and physical ability) of 51%, having received at least one prior systemic therapy (68%). With a minimal 600 mg daily (once) intake and 12 months follow-up, a 77% ORR with 24.6 months and 19 months, median DOR and mPFS, the outcomes were noted as significant. The 43% sufferers exhibiting baseline CNS had an ORR of 74% while the median DOR and PFS were 12.6 months and 13.6 months. These observations inferred a potent entrectinib efficacy, on systemic as well as in the CNS conditions. The sufferers with baseline CNS disorder exhibited an 80% ORR with 24.6 months and 26.3 months DOR and PFS. Some conclusions could not be made due to non-estimated median OS on 15.5 months follow-up. Yet again, weight gain (8%) and neutropenia (4%) were noted as significant grade 3 or 4 AE, although no deaths occurred. Readers are suggested to refer the specific source for details [130].

The NTRK prevails as a member of TRK family, comprising NTRK1, NTRK2, and NTRK3 as its fusion genes encoding for TRKA, TRKB, and TRKC respectively (Figure 2). The NTRK1 was first recognized as an oncogene in 1982 with its complementary DNA (cDNA) being isolated in 1989 [135, 136]. In 1991, TRKA expression in nervous system was unveiled, as a receptor for neutrophin nerve growth factor (NGF) with TRKB/TRKC emergence as members of the same family [137, 138].

NTRK receptors exhibit the binding susceptibility to multiple ligands, viz. NGF for NTRKA, neutrotrophin 4 (NT-4) for TRKB, and NT-3 for TRKC [139]. All known NTRKs play a decisive role in CNS functioning, including cell proliferation, migration, differentiation and apoptosis. Interestingly, the first NTRK inhibitor screening in clinical trials was not attempted till 2015, the last six years have witnessed two FDA-approved NTRK inhibitors and multiple 2nd generation drugs moving to clinical stage [140, 141]. Presently available NTRK inhibitors belong to the umbrella of TRKs, with some being active against ROS1 and ALK. These inhibitors forbid ligand-TRK interaction and concomitant TRK activation, blocking the activity of tumor cells overexpressing NTRK fusion proteins. This induces apoptosis with substantial inhibition of growing tumor cells. Two US-FDA approved NTRK inhibitors are larotectinib and entrectinib, screened via basket trials.

Larotectinib is the first approved TRK inhibitor for treating metastatic tumors harbouring NTRK gene fusion. In the year 2018, Drilon and team [140] published the larotrectinib efficacy in TRK fusion exhibiting tumors among the adults and children. This combined study constituted LOXO-TRK-14001 (NCT02122913), SCOUT (NCT02637687), and NAVIGATE (NCT02576431) phase I/II clinical trials. Overall, 55 patients (aged 4 months to 76 years) were screened, comprising 17 unique TRK fusion-positive tumors. The intake was optimized at 100 mg, twice daily for the sufferers having at least 1 m², body surface area. For children having less than 1 m² surface area, 100 mg per m² dosage was optimized. Analysis revealed a 75% ORR via unbiased central review and 80% concerning the investigator’s assessment. Over a 9.4-month median follow-up, ~86% sufferers with a response progressed with the treatment, primarily having undergone curative intent intake. Major side effects included gastrointestinal effects though 30% sufferers exhibited dizziness, the gross toxicity was grade 1 and manageable. Based on these multicentred single-arm trials, the FDA sanctioned the accelerated approval to larotectinib use for adults and children, having NTRK gene fusion-positive solid tumors, in November 2018. Results for updated efficacy and safety vide longer follow-up, were reported in the 2021 ASCO meeting, enrolling 218 patients of which 206 were evaluable for efficacy. Amongst the screened patients, 44% were NTRK1 positive, 3% for NTRK2 and 53% for NTRK3. Of the 21 distinct tumors treated, 9% belonged to the lungs. The median age was 38 years, with 45% sufferers, having received two or more, earlier systemic therapies while 27% exhibited no such history. Analysis revealed a 75% ORR with 22% complete and 53% partial respondents while 16% and 6% significant and progressive disease fates respectively. The mPFS was 35.4 months with 20.3 months follow-up. Safety evaluation on 53 patients with more than 24-month treatment did not reveal any new indications, whereby 18% of screened population had grade 3–4 AE and only 2% patients discontinued due to AEs. The observations thereby, suggested the larotrectinib medication safety [142]. The ASCO 2021 meeting updated the observations of NSCLC clinical trials NCT02576431 and NCT02122913. The entire 20 patients were heavily pre-treated with a median of 3 systemic therapies, having 48.5 years as the median age. Nineteen of screened cases had non-squamous NSCLC while one had neuroendocrine cancer, NTRK1 gene fusion was exhibited by 80% sufferers while the remaining carried NTRK3. Intake was fixed at 100 mg/m² continually till 28-day until the disease progression, withdrawal, or unbearable toxicity. The 15 evaluable sufferers exhibited an ORR of 73% with 3 having stable disease and one with progressive disease. The median response duration was 1.8 months while the median OS was 40.7 months at 16.2 months median follow-up. Major AEs belonged to grade 1 or 2 with no treatment discontinuation [143].

The second TRK inhibitor was entrectinib, an oral TKI for ROS1, ALK, and NTRK gene fusions. Inceptive approval to entrectinib was granted in June 2019 for adult and paediatric advanced NSCLC patients with NRTK fusions. In an integrated study, entrectinib displayed a 50% ORR in the NTRK fusion-positive solid tumors which improved to 70% for ROS1-positive NSCLC sufferers. Interestingly, the intracranial ORR was 55% for both ROS1 positive and NTRK fusion carrying solid tumors with 12.6–24.6 month ranged median response [144, 145]. In the past three years, STARTRK-1, STARTRK-2, ALKA-372-001 (all more than 18 years with metastatic NTRK fusion-positive tumors) and STARTRK-NG (21 years, young paediatric sufferers) trials screened entrectinib anti-NSCLC efficacy [141]. In all, 54 patients having ten distinct tumors with 19 histologies, were given a minimal single entrectinib dose. With a 12.9 month follow-up, 7% sufferers exhibited a complete response (CR) while 50% responded partially. The median response was 10 months with majority of toxicities being grade 1 or 2, mainly dizziness. The most common grade 3 or 4 AE was weight gain, noticed in 10% NTRK fusion-positive safety group and 5% in the overall safe evaluable group. Relying on these observations, the US-FDA granted accelerated approval to entrectinib for adults and paediatrics, aged 12 years or more and having solid tumors characterized by an NTRK gene fusion with no resistance to NTRK inhibitors. Approval was also given by European Medicines Agency in 2020 and in 2021, the STARTRK-2 trial demonstrating a safety profile with least treatment burden [146].

Like other TKIs, first-generation NTRK inhibitors lacked the potency to counter the mutations which affected the binding interactions, including both primary and acquired resistance [147, 148]. The 2nd generation TKIs with lower molecular weights [355.37 g/moL for repotrectinib and 380.43 g/moL for selitrectinib (DS-6051b), against 428.44 g/moL and 560.65 g/moL for larotrectinib and entrectinib] and compressed macrocyclic structures, overcome these inadequacies. Their compact structure minimizes the steric hindrance which forbids the binding of 1st generation inhibitors with the ATP binding site amidst the solvent front, gatekeeper and mutations stabilizing inactive kinase conformation (xDFG) mutations. The compact structure of 2nd generation NTRK inhibitors accommodates the resistant confirmation in distinct, TRK kinase active sites. Studies probing the larotrectinib, entrectinib, selitrectinib, and repotrectinib crystal structures in the cellular modules of TRKA/B/C fusions and resistant variants, concur these findings, in xenograft tumor models [148]. Analysis revealed the highest potency for repotrectinib for TRKA/B/C fusion with < 0.2 nmol/L half maximal inhibitory concentration (IC₅₀), succeeded by selitrectinib, entrectinib and larotrectinib with 1.8–3.9 nmol/L, 0.3–1.3 nmol/L and 23.5–49.4 nmol/L ranged IC₅₀. Repotrectinib (TPX-0005), has lately been investigated for countering ALK, ROS1 and NTRK mutations, exhibiting a more than 90 fold efficacy than crizotinib. Studies evaluating it in the ROS1 positive advanced NSCLC patients elucidated its intracranial actions. A phase I trial, comprising 65 advanced NSCLC patients having ALK, ROS1, or NTRK positivity of which, 23 had baseline CNS abnormality. Follow-up revealed dysgeusia, dizziness, paresthesia and nausea as frequent TEAEs, dose-limiting toxicity in 2 (240 mg and 160 mg) sufferers while maximum tolerated dosage could not be reached. Significant response was observed in 8 cases, with ROS1 and NTRK positivity. The ORR of 90% and 28% were noticed in 10 TKI-naive and pre-treated patients. Intake of 160 mg or more enabled a 44% response in 9 TKI pre-treated NSCLC patients, inferring below-par compatibility. Interestingly, 3 TKI naive NSCLC sufferers exhibited a 100% ORR which reduced to 50% in 4 TKI pretreated sufferers [98].

Another potent TKI having a high affinity for ROS1 and NTRK kinases is taletrectinib (DS-6051b), having significant tolerance besides an inhibitory response, in the phase I/Ib trial. Of the six patients screened for efficacy, two responded partially and developed a stable disease [149]. First-in-human, phase I results for taletrectinib were published in September 2020, involving 46 adult neuroendocrine sufferers exhibiting tumor-induced pain or ROS1/NTRK rearrangements. Inspection revealed nausea (47.8%), diarrhoea (43.5%) and vomiting (32.6%) as prominent AEs. Reduced pain scores were noticed following the 800 mg once-daily dose cohort. Substantiated ORR of 33.3% was noted in six patients with response evaluation criteria in solid tumors (RECIST)-evaluable crizotinib refractory ROS1 mutated NSCLC. This study also demonstrated a prevalence of cabozantinib sensitive ROS1 L2086F mutation as acquired resistance. The latest progress in taletrectinib evaluation is reported as a phase I study on United States and Japanaese citizens [150]. The study analysed 22 of the 61 enrolled patients, who were administered taletrectinib at 400, 600, 800, and 1,200 mg once daily via oral route. The extent was 400 mg twice daily for dose-escalation evaluation. The median follow-up time was 14.9 months, wherein 18 sufferers with ROS1 mutation were accessible for response. To a surprise, the confirmed ORR for ROS1 TKI-naive sufferers was 66.7% with a 100% DCR while the same extent for crizotinib pretreated patients was 33.3% with 88.3% control rate. Median PFS for 11 ROS1 TKI naive patients was 29.1 months while for 8 crizotinib-refractory sufferers, it was 14.2 months. The frequent AEs noted herewith included alanine transaminase and aspartate transaminase aggravations (till 72.7%), nausea and diarrhoea (till 50%). Another potent NTRK mutation specific TKI is selitrectinib (LOXO-195), currently ongoing phase I/II study (NCT03206931, NCT03215511). Some case studies have indeed highlighted the selitrectinib potent ability. One of these is a 2021 attempt wherein, a solitary patient with mammary analogue secretory carcinoma of parotid gland and ETV6-NTRK3 fusion initially responded to entrectinib (STARTRK-2 trial). The patient subsequently developed a secondary resistance to entrectinib via NTRK3 G623R mutation, thereafter responded to selitrectinib [151]. Another significant effort is a 2020 study, wherein a 47-year old woman carrying unclassified sarcoma and TPM3-NTRK1 fusion, initially attained a partial response (PR) towards larotrectinib. On developing NTRK1 G595R solvent-devoid mutation, the sufferer was switched to selitrectinib, attaining PR in 3 months. Thereafter, the patient exhibited disease progression at multiple sites, which were resected via surgery. Post-surgery resumption of selitrectinib resulted in a better response and the patient lived without any disease for more than one year, herewith [152].

The noted proto-oncogene, MET resides on chromosome 7q21–q31 and encodes for a transmembrane receptor, referred as “hepatocyte growth factor receptor”. A tyrosine kinase receptor that binds with hepatocyte growth factor (HGF), the MET gene regulates cell proliferation, survival, migration, invasion, angiogenesis and epithelial to mesenchymal transition via downstream activation of RAS/ERK/MAPK, PI3K-AKT, Wnt/β-catenin, STAT signaling pathways [156]. MET dysregulation involves sequential heterogeneous alterations, including amps and exon 14 abnormalities, extending the activation of cellular MET receptor and downstream signaling pathways [46]. Exon 14 skipping mutation (EM) of MET has been demonstrated as a discrete NSCLC hallmark, prevailing discretely from the EGFR, ALK and c-ROS mutations besides being associated with a poor prognosis. Several studies describe MET exon 14 EMs as clinically distinct NSCLC subtype, addressed via patient stratification-driven personalized therapy [157, 158].

Abnormalities in MET gene expression are noticed in less than 5% of NSCLC sufferers. Usually, MET amp is ascertained via fluorescence in situ hybridization (FISH) and next generation DNA or RNA sequencing. The last two decades have witnessed multiple MET targeting agents comprising multikinase inhibitors (crizotinib, cabozantinib, MGCD265, AMG208, altiratinib, golvatinib), selective inhibitors (capmatinib, tepotinib, tivantinib) and monoclonal antibodies (mAbs; onartuzumab, emibetuzumab, ficlatuzumab, rilotumumab). Crizotinib, cabozantinib, tepotinib, capmatinib, amivantamab and savolitinib are the noted CDs evaluated for MET mutated anti-NSCLC therapy [159, 160]. Crizotinib is a multi-targeted small-molecule TKI, majorly targeting the ALK, ROS1, and MET genes. Clinical potency of crizotinib against MET exon 14 skipping harboring NSCLC is notified in selective cases. A phase I PROFILE 1001 study on 69 sufferers was attempted by Shaw and teammamtes [112], wherein crizotinib treatment (as singular agent) was screened for 7.3 months (mPFS), reaching a 32% ORR. Tepotinib prevails as an exclusive MET selective inhibitor, which disrupts the MET signal transduction for its potent anti-NSCLC activity [161]. Screening the tepotinib efficacy in exon 14 altered NSCLC patients, a phase II trial (VISION) by Paik and associates [162] observed a significant response via 500 mg daily administration in 152 patients. Follow-up of 99 of these sufferers till a minimum of nine months, revealed tepotinib association with PR in ~50% sufferers, with a 46% ORR (95% confidence), monitored via an independent review and 56% investigator assessment. The PFS and OS were 8.5 months and 17.1 months. The findings awarded regulatory approval to tepotinib in MET exon 14 EMs, in March 2020 in Japan [162].

Capmatinib is another selective MET inhibitor, which impairs the downstream effector’s activation in the MET signaling pathway by hindering MET phosphorylation. Screening the capamitinab efficacy via a phase II study, involving MET exon 14 skipping NSCLC patients (assigned to cohorts as per the previous lines of therapy), Wolf and associates [163] examined the sufferers having received one or two lines of therapy. The investigators noted that a 400 mg capmatinib tablet intake twice daily, exhibited a 41% ORR and a mean PFS of 52 months. The extents were 68% (ORR) and 12.4 months (PFS) in treatment naive sufferers [163]. Yet another phase II study screened capmatinib efficacy in 97 sufferers via post-therapy screening and noticed an ORR of 31.9% in pretreated patients whereas treatment naive sufferers exhibited a response of 71.4%. Familiar AEs in both categories were peripheral edema, nausea, and vomiting [164]. Savolitinib is a MET receptor inhibitor that specifically inhibits MET activation by disrupting the MET signal transduction in an adenosine triphosphate competitive regime, whereby it inhibits the tumor cell growth [165]. In a recent trend, Lu and accomplices [166] performed a multicenter phase II study to screen the savolitinib efficacy, corresponding to 600 mg and 400 mg in Chinese NSCLC (exon 14 altered) patients. Twenty-five of the examined sufferers had pulmonary sarcomatoid carcinoma (PSC), a rare aggressive form of NSCLC while 45 others exhibited NSCLC histology. The primary endpoint as ORR was monitored in tumor response evaluable (61 patients) while the sensitivity screening was done in all screened 70 patients. Sufferers of both groups exhibited significant ORR (49% in an evaluable group having MET exon 14 mutated NSCLC and 42.9% in the full analysis set), both with 95% confidence limits. Savoliyinib provided significant survival betterment irrespective of tumor subtypes with more than 40% ORR of each group as well as in treatment-naive patients. Significantly though, savolitinib also proved significant in arresting brain metastasis of tumors [166]. The studies discussed, thereby suggest a superior efficacy of MET selective TKIs to treat exon 14 skipping mutated NSCLCs than the non-selective TKIs. It summarizes the clinical trials evaluating the MET inhibitors specifically targeting the exon 14 EM for NSCLC manifestation (Table 3) [162, 163, 166, 167].

Amivantamab is a prominent antibody, known for its EGFR and MET-targeting specific actions [168]. Multiple in vitro and in vivo studies have demonstrated the amivantamab’s ability to inhibit the EGFR and MET signaling via ligand blocking and biochemical degradation. Noted amivantamab actions, include its ability to induce trogocytosis and allocate immune effector cells for eliminating EGFR and MET expressing tumor cells, vis-à-vis antibody-dependent cellular cytotoxicity [168, 169]. In a phase I study involving metastatic NSCLC, amivantamab was screened for varied tumor subgroups, comprising EGFR exon 20 insertions, MET exon 14 and MET amp mutations. Though MET mutation-specific results are not yet reported, the 40% ORR towards EGFR exon 20 insertions led to the amivantamab FDA approval for EGFR exon 20 insertions in NSCLC patients after platinum chemotherapy progression [169]. It would be significant to screen the efficacy of remaining cohorts, wherein amivantamab expansion to counter MET exon 14 mutation and MET amp NSCLC could be validated.

Rearrangement during transfection (RET) gene exerts via RTK, a proto-oncogene located on chromosome 10 (10q11.2), encoding for RET protein. RET rearrangements account for 1–2% NSCLC adversities (majorly LUAD) and prevail in a mutually exclusive regime to EGFR, ALK, ROS1, and KRAS mutations [183]. To date, most frequent partner of RET rearrangements in NSCLC is kinesin family member 5B (KIF5B), prevailing as the KIF5B-RET fusion gene, with a higher frequency in young non-smokers [184, 185]. Selpercatinib is the first FDA-approved RET inhibitor for NSCLC treatment [186]. Bevacizumab [targets vascular endothelial growth factor (VEGF) in advanced non-squamous NSCLC] and several multi-kinase inhibitors (cabozantinib, vandetanib, lenvatinib and sunitinib) are being evaluated for their anti-VEGF activities. The VEGF is critically implicated in angiogenesis which further aggravates the metastasis and tumor cell invasion. Cabozantinib and vandetanib are the prominently screened RET inhibitors, wherein a 20–50% response has been observed in the RET rearranged NSCLC sufferers [187, 188]. With a 16–20% response rate, lenvatinib and sunitinib occupy the second place but both amiss the RET targeting activity [189, 190].

Insignificant actions of multi-targeted TKIs in RET-rearrangements characterized NSCLC, have gradually switched the attention to the next generation, RET-selective inhibitors, such as RXDX-105, BLU-667, and LOXO-292. RXDX-105 is a VEGF receptor (VEGFR)-sparing RET inhibitor, with a phase Ib study on 21 untreated RET fusion-positive NSCLC sufferers, observed response in 6 of the 8, non-KIF5B-RET fusion positive patients. Unfortunately, none of the 13 patients carrying KIF5B-RET fusions met the prescribed response evaluation criteria for solid tumors [191]. Evaluation of BLU-667 exhibited a ten-fold higher efficacy than FDA approved anti-RET (oncogenic rearrangements) MKIs. Investigation via phase I study revealed an objective response in 50% (7 of the 14) BLU-667 administered RET-fusion carrying NSCLC sufferers [192]. In a more recent attempt, 79 advanced LC subjects harboring RET fusion rearrangements were administered BLU667. Analysis revealed a 56% ORR in 57 screened patients, with a 91% DCR. Six of the screened patients exhibited a > 6-month response with hypertension, constipation, increased aspartate aminotransferase (AST) and alanine aminotransferase (ALT), and reduced neutrophil count, as major TEAEs [193]. LOXO-292 is another RET-selective TKI, whose efficacy towards RET-driven cancers, was evaluated via phase I basket trial, at the American Society of Clinical Oncology Annual Meeting in 2018. In all, 27 NSCLC patients were recruited, 17 of which attained an evaluable ORR (one withdrew), with a predicted response time of > 6 months [194]. Of note, each of the early phase trials (on RXDX-105, BLU-667 & LOXO-292 exhibited the CNS penetration to a certain extent.

A phase I/II, ARROW, multi-cohort study evaluated pralsetinib on RET fusion-positive 281 NSCLC patients, aged > 18 years. Yester success via the oral route apart from CNS penetration, selective RET inhibition, and clinical efficacy in RET fusion harboring NSCLC sufferers were the encouragements to pursue the studies. Inspection revealed an ORR of 72% in treatment naive patients while for prior platinum chemotherapy administered sufferers, the extent was 59% (both estimates made at 95% CI). While the median response duration for treatment naive sufferers could not be reached, it was 22.3 months for platinum chemotherapy-administered patients. Inspection revealed tumor shrinkage in all treatment naive sufferers while in platinum chemotherapy-administered patients, the extent was 97%; the mPFS was 13 months and 16.5 months respectively. The intracranial response rate in the sufferers with measurable intracranial metastasis was 70%, with all having received prior systemic treatment. Noted AEs in treatment naive sufferers included neutropenia (18%), hypertension (10%), increased blood creatine phosphokinase and lymphopenia (9% each). The findings validated pralsetinib efficacy with tolerable response in treatment-naive, RET fusion-positive advanced NSCLC sufferers. Phase III trial (NCT04222972), evaluating pralsetinib and standard approach in first-line setting is currently awaiting a response [195].

This mutation is noticed in 1–3% NSCLC patients with a history of smoking and the altered BRAF gene, forms a non-native BRAF protein that fuels the tumor cell growth. Dabrafenib (Tafinlar) and trametinib (Mekinist), respectively the BRAF and MEK inhibitors, are used to ramify these mutations. A typical dabrafenib and trametinib combination was approved for the treatment of BRAF V600E mutation exhibiting NSCLC, based on the results of an open-label trial. Of the 93 enrolled patients, 57 had received a prior systemic treatment while 36 were naive. Each patient received 150 mg dabrafenib orally twice (on a daily mode) and 2 mg trametinib, OD. Analysis revealed 63% and 61% ORR in pretreated and treatment naive sufferers. The corresponding response duration was ≥ 6 months for 64% previously treated and 59% treatment naive sufferers [196, 197].

Immune checkpoints are the co-stimulatory and co-inhibitory receptors that help in the priming and activation of T cells. Most extensively studied immune checkpoint receptors for cancer therapy are cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and PD-1. PD-1 is an immunological receptor from the CD28/CTLA-4 family that inhibits the antigen receptor signaling via localizing the protein tyrosine phosphatase, SHP2 on interacting with its ligands, PD-L1 or PD-L2. Owing to significant ligand diversity, this receptor is involved in manifold immune responses.

Therapies targeting PD-1 are programmed to inhibit the ligand binding with the receptor (PD-L1 inhibitors), generating the requisite immune response against the tumor cells [200, 201]. Numerous cells express ligands that bind these receptors and block the immune response to self-antigens under apt physiological circumstances [202]. Many tumor cells use this mechanism to evade immunosurveillance by ligand bound state of these receptors [203]. By obstructing these inhibitory pathways, the ICIs activate the T-cells against tumor cells to kill the tumor cells [204]. Antibodies targeting immune checkpoints result in enhanced immunologically triggered tumor cell death. Following paragraphs briefly summarize the recent studies using CTLA-4 and PD-L1 inhibitors for NSCLC treatment.

Cytokines are the small secretory proteins produced by immune (majorly) and non-immune cells, serving as molecular messengers for interacting with other cells. Their decisive roles in tumor aggravation are characterized by regulatory participation in cancer immunity cycle, including cancer antigen presentation, T cell priming and activation, effector T cell infiltration to the tumor location and cancer cell death (Figure 5). ILs are the most important cytokines, aggravating tumor growth via multiple mechanisms.

Notably, IL-1α acts as an “alarmin”, mediating the initial stages of sterile inflammation besides recruiting neutrophils and monocytes in response to tissue injury [228]. In malignancies, IL-1α activates the TAMs which in turn, releases multiple growth factors and cytokines that aggravate the tumor cell proliferation, angiogenesis besides suppressing the adaptive immune response [229]. In a phase I study, a human MAb targeted towards IL-1, mammalian actin binding protein 1 (MABP1) was administered to 16, chemotherapy non-responsive NSCLC sufferers, in a dose-dependent manner. Analysis revealed a 7.6 months median OS with a 57 days, PFS. The treatment efficacy exhibited a correlation with anti-EGFR pretreated patients (9.4 months median OS), who responded better than others (4.8 months OS). The observations were supported by higher mPFS (97 days) in the anti-EGFR pretreated sufferers than others (PFS: 78 days). A small size was the lacuna in this study, making it inconclusive [230]. Another cytokine, IL-2 was first described as an immuno-stimulatory factor for the expansion of activated effector T cells, but it was later described as an immuno-suppressor based on doses [231]. A phase III randomized multicenter trial screened the impact of subcutaneous low-dose IL-2 with standard chemotherapy, on OS in NSCLC patients. Surprisingly, there were no discernible differences in outcomes on the inclusion of IL-2 to chemotherapy [232].

Lately, the association of some characteristic mutated gene expressions have been reported distinctly for the success of immunotherapy. This section discusses a few recent attempts (mostly from 2022) reporting a comparatively higher efficacy with characteristic gene mutations prevailing natively or amidst chemotherapy. The first study of note is an attempt by Liu and colleagues [250] who noted KRAS-G12D mutation responsible for suppressing the PD-L1 targeted therapy and CXCL10/CXCL11 (chemokines) secretion, together suppressing the CD8⁺ tumor infiltrating lymphocytes (TILs). Analyzing the KRAS-G12D mutation exhibiting cell lines, the investigators noted a corresponding suppressed extent of PD-L1 via 70 kDa ribosomal protein S6 kinase (P70S6K)/PI3K/AKT axis and suppressed CXCL10/CXCL11 levels by inhibiting the high mobility group protein A2 (HMGA2) expression levels. The observation was counter-screened by co-delivering PD-L1 blockade with paclitaxel (alone being an HMGA2 upregulation and concomitant stimulation of CXCL10/CXCL11), revealing a decreased tumor growth compared to singular treatment with a PD-L1 inhibitor, in the KRAS-G12D-mutant LUAD carrying mouse model. Screening the distinction in the co-delivery mode, it was noticed that paclitaxel with PD-L1 inhibitor significantly engaged the CD8⁺ TILs towards enhanced CXCL10/CXCL11 expression extents. The study, therefore, clarified the mechanism by which KRAS mutant NSCLC exhibit resistance to immunotherapy, noticing a better response of paclitaxel + ICI-mediated treatment (than only ICIs) in KRAS-G12D mutant NSCLC sufferers [250].

A nearly similar study by Landre and associates [251] examined IMpower-150, KEYNOTE-189, and KEYNOTE-042 first-line and OAK, POPLAR, CHECKMATE 057, second-line clinical trials. These trails together comprised 386 KRAS mutant and 927 KRAS wild-type tumors. Analysis revealed anti-PD-L1 treatment with or without chemotherapy in KRAS mutant NSCLC sufferers to exhibit an enhanced OS (0.59) and PFS (0.58), compared to singular chemotherapy, in both clinical trials. A striking coincidence was the observation of enhanced OS and PFS only in KRAS mutant NSCLC but not in KRAS wild-type tumors. Thereby, the results explained a better response of singular and chemotherapy combined anti-PD-L1 therapies, for KRAS mutant and wild-type NSCLCs, with a higher OS for the mutant tumors [251]. Interesting mutational correlations were also demonstrated by Ricciuti and associates [252], who screened the cohort of 1,552, PD-1/PD-L1 blockade-received NSCLC patients, of which 830 were females and 1,347 had non-squamous histology. Analysis revealed a 57% ORR for PD-L1 inhibition in the sufferers having high tumor mutational burden (TMB) and 50% (or greater) PD-L1 expression and a mere 8.7% for those having low TMB and < 1% PD-L1 expression. The observations led the investigators to correlate the TMB expression extents with immune cell infiltration and inflammation-mediated T-cell mediated response, together enhancing the sensitivity to PD-1/PD-L1 blockade in the NSCLC cells expressing PD-L1 [252].

Combinatorial therapies have been the choice in the recent past to overcome the eventual resistant outcomes in monotherapeutic programmed schedules. For checkpoint inhibitors (CKIs), this realization gathers support from the likely additive or synergistic outcomes via enhancing the blockade of immune cells inhibition, using 2 distinct CKIs. The salient observations of such attempts employing PD-L1 and CTLA-4 pathway-targeting agents are summarized in the following paragraphs.