Review of Therapeutic Strategies for Anaplastic Lymphoma Kinase-Rearranged Non-Small Cell Lung Cancer

Simple Summary Anaplastic lymphoma kinase (ALK)-rearranged non-small cell lung cancer (NSCLC) was first reported in 2007. Following the development of crizotinib as a tyrosine kinase inhibitor (TKI) targeting ALK, the treatment of advanced NSCLC with ALK-rearrangements has made remarkable progress. Currently, there are five ALK-TKIs approved by the FDA, and the development of new agents, including fourth-generation TKI, is ongoing. Clinical trials with angiogenesis inhibitors and immune checkpoint inhibitors are also underway, and further progress in the treatment of ALK-rearranged advanced NSCLC is expected. The purpose of this manuscript is to provide information on the recent clinical trials of ALK-TKIs, angiogenesis inhibitors, immune checkpoint inhibitors, and chemotherapy, to describe tissue and liquid biopsy as a method to investigate the mechanisms of resistance against ALK-TKIs and suggest a proposed treatment algorithm. Abstract Non-small cell lung cancer (NSCLC) with anaplastic lymphoma kinase rearrangement (ALK) was first reported in 2007. ALK-rearranged NSCLC accounts for about 3–8% of NSCLC. The first-line therapy for ALK-rearranged advanced NSCLC is tyrosine kinase inhibitors (TKI) targeting ALK. Following the development of crizotinib, the first ALK-TKI, patient prognosis has been greatly improved. Currently, five TKIs are approved by the FDA. In addition, clinical trials of the novel TKI, ensartinib, and fourth-generation ALK-TKI for compound ALK mutation are ongoing. Treatment with angiogenesis inhibitors and immune checkpoint inhibitors is also being studied. However, as the disease progresses, cancers tend to develop resistance mechanisms. In addition to ALK mutations, other mechanisms, including the activation of bypass signaling pathways and histological transformation, cause resistance, and the identification of these mechanisms is important in selecting subsequent therapy. Studies on tissue and liquid biopsy have been reported and are expected to be useful tools for identifying resistance mechanisms. The purpose of this manuscript is to provide information on the recent clinical trials of ALK-TKIs, angiogenesis inhibitors, immune checkpoint inhibitors, and chemotherapy to describe tissue and liquid biopsy as a method to investigate the mechanisms of resistance against ALK-TKIs and suggest a proposed treatment algorithm.


Introduction
Anaplastic lymphoma kinase (ALK) was first discovered as a fusion partner in the (2;5) chromosomal translocation in anaplastic large cell lymphoma in 1994 by Morris et al. [1]. ALK is a transmembrane tyrosine kinase encoded by the ALK gene localized on chromosome 2, belonging to the superfamily of insulin receptors. ALK expression is considered to be involved in the development of the nervous system [2]. ALK regulates several pathways involved in cell survival, proliferation, and cell cycling, including the AKT/PI3K [3] and STAT3 pathways [4,5]. The echinoderm microtubule-associated protein-like-4 (EML4)-ALK

Diagnosis
International guidelines recommend testing for ALK mutations in all non-squamous NSCLC [12,13]. There are several methods to detect ALK-rearrangement: fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), and polymerase chain reaction (PCR)-based next-generation sequencing (NGS). The choice of which test to use, and the algorithm is controversial and is an issue for consideration. The advantage of NGS is that it can be used in liquid biopsy using circulating tumor cells (ctDNA), etc., not only tissue samples, and it can detect drug resistance mechanisms. Identification of the resistance mechanism to ALK-TKIs and corresponding treatment are expected to improve prognoses. Liquid biopsy is aimed at predicting drug resistance mechanisms and treatment efficacy using specimens such as ctDNA, cell-free DNA (cfDNA), and circulating tumor DNA in the blood. The validity of ALK-rearrangement detection by liquid biopsy using NGS was demonstrated in the Blood First Assay Screening Trial (BFAST) [14] and the NILE (Noninvasive versus Invasive Lung Evaluation) study [15]. In the BFAST study, alectinib

Diagnosis
International guidelines recommend testing for ALK mutations in all non-squamous NSCLC [12,13]. There are several methods to detect ALK-rearrangement: fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), and polymerase chain reaction (PCR)-based next-generation sequencing (NGS). The choice of which test to use, and the algorithm is controversial and is an issue for consideration. The advantage of NGS is that it can be used in liquid biopsy using circulating tumor cells (ctDNA), etc., not only tissue samples, and it can detect drug resistance mechanisms. Identification of the resistance mechanism to ALK-TKIs and corresponding treatment are expected to improve prognoses. Liquid biopsy is aimed at predicting drug resistance mechanisms and treatment efficacy using specimens such as ctDNA, cell-free DNA (cfDNA), and circulating tumor DNA in the blood. The validity of ALK-rearrangement detection by liquid biopsy using NGS was demonstrated in the Blood First Assay Screening Trial (BFAST) [14] and the NILE (Noninvasive versus Invasive Lung Evaluation) study [15]. In the BFAST study, alectinib was shown to have a non-inferior outcome in patients with ALK-rearranged NSCLC diagnosed by blood-based NGS compared to those diagnosed by tissue-based NGS. The NILE study showed that cfDNA analysis using Guardant360 was non-inferior to tissue samples in detecting guideline-recommended biomarkers. These results demonstrated that for patients who, for some reason, do not have ALK-rearrangement identified in tissue samples, there is an opportunity to treat with ALK-TKIs based on blood-based NGS results. However, it has been reported that the level of detection of ALK-rearrangements by liquid biopsy is lower than that by tissue biopsy due to the small amount of acid released into the blood in small or slow-growing tumors, etc. [16]. Tissue biopsy and liquid biopsy have their own advantages and disadvantages. Tissue rebiopsy has advantages in histological evaluation, allowing for the evaluation of EMT transformation and gene amplification. Biopsies, however, are highly invasive and expensive because of the need for bronchoscopy or surgery. Liquid biopsy is not capable of histological evaluation but is non-invasive and is therefore repeatable and cost-effective. A liquid biopsy may be particularly useful when tissue samples are insufficient or when tissue biopsy is contraindicated. Detecting driver mutations and ALK resistance mutations as therapeutic targets is very beneficial, even when sufficient tissue samples are not available for biopsy. In addition, liquid biopsy is easily repeatable, allowing for real-time, long-term monitoring during treatment and the early detection of recurrence before clinical symptoms appear [17]. Although liquid biopsy for ALK-rearranged NSCLC is still under investigation, it is expected to be a useful tool for detecting genetic mutations with false-negative results by tissue biopsy and investigating resistance mechanisms.

Intracranial Efficacy
It has been reported that crizotinib has low penetration of the blood-brain barrier (BBB) [24,25]. Lower cerebrospinal fluid (CSF) concentrations and CSF/plasma ratios prevent the achievement of therapeutic concentrations in the brain and lead to pharmacological tolerance. In the PROFILE 1014 study, the incidence of extracranial PD only was less with crizotinib than with chemotherapy, regardless of the presence or absence of brain metastasis (BM) prior to treatment initiation (ITT population: 73% vs. 80%, BM present: 57% vs. 60%, BM absent: 78% vs. 86%). In contrast, the proportion of patients for whom the brain was the only site of PD was higher with crizotinib than with chemotherapy (ITT population: 24% vs. 10%, BM present: 38% vs. 23%, BM absent: 19% vs. 6%). In contrast to crizotinib, the next-generation ALK-TKIs passed through the BBB and had higher concentrations in the CSF. The clinical trial comparing crizotinib with the next-generation ALK-TKIs showed that crizotinib had inferior activity in the CNS.
Recently, crizotinib has been used less frequently due to its inferior intracranial efficacy and shorter mPFS compared to next-generation ALK-TKIs.

Intracranial Efficacy
In the ASCEND-4 study, intracranial ORR in patients with measurable brain metastases at baseline was 72.7% (CR: 2, PR: 14) in the ceritinib arm (n = 22) and 27.3% (CR: 2, PR: 4) in the chemotherapy arm (n = 22). The median intracranial duration of intracranial response (IC-DOR) was 16.6 months (95% CI: 8.1-NE) in the ceritinib arm. The median IC-DOR could not be estimated in the chemotherapy arm because four of six patients had not progressed at analysis.
Despite these efforts to counteract adverse events, ceritinib is still used less often than other ALK-TKIs due to gastrointestinal toxicity issues and the fact that crizotinib is not the control arm in the ASCEND-4 study, which led to its approval for first-line therapy.

Intracranial Efficacy
In the ALEX trial, alectinib was reported to have a better intracranial response compared to crizotinib. The CNS ORR in patients with measurable CNS metastases at baseline was 85.7% in the alectinib arm versus 71.4% in the crizotinib arm in patients with previous radiotherapy and 78.6% in alectinib arm versus 40.0% in crizotinib arm in patients without previous radiotherapy. In patients with measurable/not measurable baseline CNS metastases, CNS DOR was NR (95% CI: 14.8-NR) in the alectinib arm and 11.1 months (95% CI: 13.7-18.1) in the crizotinib arm in patients with prior radiotherapy. The CNS DOR in patients without previous radiotherapy was NR (95% CI: 13.4-NR) in the alectinib arm and 3.7 months (95% CI: 2.3-5.5) in the crizotinib arm, which was longer than that for crizotinib.
Lorlatinib has been an ALK-TKI frequently used after first-line treatment but based on the results of the phase III CROWN trial, lorlatinib is expected to be used more frequently in first-line treatment. Whether it is better to use lorlatinib after second-generation ALK-TKI treatment or in the first-line setting requires further study.

Intracranial Efficacy
In the phase I/II trial, 14 patients had CNS-targeted lesions at baseline. ORR of 64.3% (95% CI: 38.8-83.7) was reported with two patients with CR and seven patients with PR. In the eXalt3 study, intracranial ORR in patients with measurable brain metastases was 54% for ensartinib and 19% for crizotinib. In patients with intracranial disease, ORR was 64% for ensartinib and 21% for crizotinib, delaying the incidence of new central lesions in patients without baseline brain metastases (23.9 months vs. 4.2 months, HR: 0.32 (95% CI: 0.15-0.64)).
The summary of six TKIs is in the tables below (Tables 1-4). ORR: overall response rate, mPFS: median progression-free survival, median overall survival.  RT: radiation therapy, IC-ORR: intracranial overall response rate, IC-DOR: intracranial duration of response.

Combination Therapy with Angiogenesis Inhibitors and ALK-TKIs
Angiogenesis inhibitors prevent tumor growth by blocking the signals that promote tumor angiogenesis. There are no clinical trials evaluating the efficacy of angiogenesis inhibitors monotherapy in ALK-rearranged NSCLC. The efficacy of ramucirumab plus erlotinib in EGFR-mutated advanced NSCLC has been reported [50]. Similarly, combination therapy with angiogenesis inhibitors and ALK-TKIs has been studied. A single-arm, prospective observational study investigating the efficacy and safety of bevacizumab plus crizotinib in ALK/ROS-1/c-MET-positive advanced NSCLC has been reported [51]. Patients received crizotinib (250 mg twice daily) and bevacizumab (7.5 mg/kg every 3 weeks) until disease progression or intolerable toxicity or death. The primary endpoint was mPFS, and the secondary endpoints were DOR, ORR, disease control rate (DCR), and safety. In 12 patients with ALK-rearranged NSCLC, mPFS was 13.9 months, and median DOR was 14.8 months. Median OS was NR, and the 3-year survival rate was 79.5%. Of the 12 patients, the best overall response was PR in seven and SD in five. ORR and DCR were 58.3% and 100%, respectively. The most common adverse events were fatigue (28.6%) and rash (21.4%). Other adverse events reported were nausea (14.3%), vomiting (7.1%), edema (7.1%), and pain (7.1%). Most adverse events (86.7%) were grade 1-2, but two patients had grade 3 or 4 elevations in aminotransferases, and both discontinued treatment. In addition, one patient reported grade 1 hemoptysis, and treatment was discontinued. No interstitial lung disease, active bleeding, hypertension, or treatment-related death occurred. Though this study initially planned to enroll 30 or more patients, however, during the enrollment, second-generation ALK inhibitors were approved as first-line treatment, which made the enrollment difficult, and the sample size was small. However, several other combination therapies with angiogenesis inhibitors and ALK-TKIs are also ongoing (NCT:03779191, NCT:02521051: bevacizumab plus alectinib), (NCT:04227028: bevacizumab plus brigatinib), (NCT:04837716: carboplatin, pemetrexed, bevacizumab plus ensartinib).

Immune Checkpoint Inhibitor
An immune checkpoint inhibitor (IO) that targets programmed cell death-1 (PD-1), programmed cell death ligand-1 (PD-L1), and CTLA-4 is an important therapeutic agent for NSCLC. The presence of EML4-ALK is associated with increased PD-L1 expression by activating the PI3K-AKT and MEK-ERK pathways [52]. Therefore, treatment of ALKrearranged NSCLC with immune checkpoint inhibitors is expected to be an effective therapeutic strategy. However, IO has been reported to lack efficacy in NSCLC with oncogenic driver mutations, such as EGFR and ALK. In a single-center retrospective study evaluating NSCLC patients with EGFR-mutant (n = 22) or ALK-rearranged (n = 6) NSCLC treated with IO, objective response was observed in only one patient with EGFR mutation and ORR in patients with ALK-rearrangement was 0%, in contrast to 7 of 30 patients (23.3%) with EGFR wild type or ALK-negative/unknown (p = 0.053) [53]. Another multicenter retrospective study reported an ORR of 0%, mPFS of 2.5 months (95% CI: 1.5-3.7), and mOS of 17.0 months (95% CI: 3.6-NR) for 23 NSCLC patients with ALK-rearrangement treated with IO monotherapy [54]. In a retrospective real-world study of 83 patients (of these, 74 patients received IO as monotherapy) with ALK-rearranged NSCLC who received IO from a multicenter electronic medical record-derived database, the mPFS of patients who received IO before ALK-TKI was 3.9 months, and that of patients who received IO after ALK-TKI was 1.5 months [55]. CheckMate 057 [56] and KEYNOTE-010 [57] are prospective clinical trials treating NSCLC patients with IO, including ALK-rearranged NSCLC patients. However, due to the small number of patients in both trials, outcomes for the ALK-positive cohort have not been reported. Combination therapy with ALK-TKIs and IO has been evaluated in several trials, but many trials have resulted in poor efficacy and increased toxicity. Group E of the phase I/II CheckMate 370 evaluated the safety of the combination of nivolumab (240 mg once every two weeks) and crizotinib (250 mg twice daily) as first-line treatment for previously untreated ALK-rearranged advanced NSCLC patients. Of the 13 patients, 5 (38%) developed severe hepatotoxicity and discontinued the combination therapy, and 2 of the 5 patients died. ORR was reported to be 38% [58]. In the phase Ib study [59] of pembrolizumab plus crizotinib for previously untreated ALKrearranged advanced NSCLC, one of the first two patients enrolled at dose level 0 (crizotinib 250 mg twice daily and pembrolizumab 200 mg every 3 weeks) required discontinuation of pembrolizumab due to grade 3 liver toxicity, and one died of grade 4 pneumonia induced by pembrolizumab. At dose level -1 (3 weeks of crizotinib monotherapy 250 mg twice daily, followed by crizotinib 250 mg twice daily, with the addition of pembrolizumab 200 mg every 3 weeks), two of the seven patients required pembrolizumab discontinuation and crizotinib discontinuation/reduction due to liver toxicity. This trial was terminated early in the dose-finding phase due to the severe adverse events identified. The phase Ib study evaluated nivolumab (3 mg/kg every 2 weeks) in combination with ceritinib (n = 14: 450 mg/day or n = 22: 300 mg/day) in 36 patients with previously treated or untreated stage IIIB/IV ALK-rearranged NSCLC [60]. In the dose-escalation study, ORR for patients without prior treatment with ALK-TKI was 83% (95% CI: 35.9-99.6) in the 450 mg/day ceritinib group and 60% (95% CI: 26.2-87.8) in the 300 mg/day ceritinib group, and ORR for patients with previous treatment with ALK-TKI was 50% (95% CI: 15.7-84.3) in the 450 mg/day group and 25% (95% CI: 5.5-57.2) in the 300 mg/day group. Elevated alanine aminotransferase level (25%), elevated gamma-glutamyl transferase level (22%), elevated amylase level (14%), elevated lipase level (11%), and maculopapular rash (11%) were reported to be common grade 3-4 adverse events. The protocol had to be changed for toxicity management, and eventually, the registration was terminated. A phase I trial of erlotinib or crizotinib in combination with the CTLA-4 inhibitor ipilimumab was conducted in patients with EGFR-mutant or ALK-rearranged NSCLC [61]. Median PFS for the three patients with ALK-rearranged NSCLC was 24.1 months. One of the three patients developed dropsy, and one developed grade 2 pneumonia, and the trial was terminated early. Trials of alectinib plus atezolizumab (NCT:02013219) have been completed, and results are awaited. Two clinical trials of combination therapy of platinumdoublet with IO (+/− angiogenesis inhibitor) for EGFR-mutant/ALK-rearranged NSCLC are ongoing (NCT:04042558: platinum-pemetrexed-atezolizumab +/− bevacizumab), along with the trial of (NCT:03991403: atezolizumab + bevacizumab + carboplatin + paclitaxel). The results of IO monotherapy and the combination of IO + TKI are summarized in Tables 5 and 6, respectively.

Chemotherapy
In a previously reported study, pemetrexed has been reported to be effective for ALKrearranged NSCLC. In the PROFILE 1007 study, median PFS in the chemotherapy arm was 4.2 months in the pemetrexed arm and 2.6 months in the docetaxel arm, suggesting that pemetrexed may be more effective than docetaxel. In addition, two retrospective studies showed that pemetrexed prolongs PFS in ALK-rearranged NSCLC. In a retrospective study of 89 advanced-NSCLC patients (ALK-positive: 19, EGFR-mutant: 12, KRAS-mutant: 21, and wild type: 37), mPFS was reported as 9 months (95% CI: 3-12) in the ALK-positive, 5.5 months (95% CI: 1-9) in the EGFR mutant, 7 months (95% CI: 1.5-10) in the KRAS mutant, and 4 months (95% CI: 3-5) in the wild type. In the multivariate analysis in this study, ALK was the only driver mutation associated with prolonged PFS in the chemotherapy regimen, including pemetrexed (HR: 0.36 (95% CI: 0.17-0.73), p = 0.0051) [62]. A retrospective study of 95 patients with advanced NSCLC (ALK-positive: 43, EGFR-mutant: 15, wild-type: 37) reported the efficacy of pemetrexed [63]. ORR was 46.7% in ALK-positive, 16.2% in the EGFR mutant, and 4.7% in the KRAS mutant. Time to progression was 9.2 months in the ALK-positive, 1.4 months in the EGFR mutant, and 2.9 months in the KRAS mutant, regardless of treatment line. ALK-positive was shown to be a significant predictor of ORR (HR: 0.07 (95% CI: 0.01-0.32), p = 0.001) and time to progression (HR: 0.44 (95% CI: 0.24-0.80), p = 0.007). Pemetrexed is an important therapeutic agent for ALK-rearranged NSCLC that is not responsive to ALK-TKIs or cannot tolerate adverse events. The efficacy of combination therapy with EGFR-TKI and platinum doublet therapy for EGFR-mutant NSCLC has been reported in two phase III trials [64,65]. Similarly, the combination of pemetrexed and ALK-TKI is well tolerated; it may improve PFS and OS. The phase II trial to evaluate the efficacy of combination therapy of platinum doublet and ALK-TKI in ALK-rearranged NSCLC is ongoing in Japan (jRCTs041210103). Recent and ongoing clinical trials are summarized in Table 7.

Drug Sensitivity
Many ALK fusion partners, such as EML4, PM-3/-4, CLTC, LMNA, PRKAR1A, RANBP2, TFG, FN1, and KIF5B, have been identified [66,67]. It is known that the activity of ALK-TKIs varies depending on the partner; for instance, KIF5B-ALK was highly sensitive to ensartinib but was one of the least sensitive fusions to crizotinib and lorlatinib [68]. In addition, several EML4-ALK variants have been reported [69]. The most frequent variant is variant 1, in which exon 13 of EML4 is fused to exon 20 of ALK (E13; A20), and the next most frequent variant is variant 3a/b, in which exon 6a or 6b of EML4 is fused to exon 20 of ALK (E6a/b; A20). Variant 3 is known to have a shorter mPFS than variant 1 with treatment with crizotinib, alectinib, and ceritinib [70,71]. The ALTA-1L trial evaluated the efficacy of each variant of brigatinib, and similarly, variant 3 showed poorer outcomes compared to variant 1 [43]. The poor prognosis of variant 3 has been attributed to the shorter variant being more stable, accumulating in greater numbers, and interacting better with the cytoskeleton, causing stronger oncogenic signaling, less sensitivity to ALK-TKIs, and accelerated migration and metastasis [72][73][74]. In a retrospective study (n = 129), resistance mutations were identified in 10 patients (30%) in variant 1 and 25 patients (57%) in variant 3 (p = 0.023). In particular, the G1202R mutation was detected in 32% (14/44) in variant 3 compared to 0% (0/33) in variant 1 (p < 0.001). In the same study, an analysis of 12 patients with variant 1 and 17 patients with variant 3, who received lorlatinib after treatment failure with both crizotinib and at least one second-generation ALK-TKI, reported that patients with variant 3 had a significantly longer PFS than patients with variant 1 (mPFS: 11.0 months vs. 3.3 months, HR: 0.31 (95% CI: 0.12-0.79), p = 0.011) [75]. The higher drug sensitivity of variant 3 to lorlatinib than variant 1 is because ALK mutations, including the G1202R mutation, which is effective for lorlatinib, are more likely to be associated with variant 3. The TP53 mutation is a common gene mutation responsible for ALK-TKI resistance [76]. A retrospective study showed that the presence of the TP53 mutation reduced sensitivity to ALK-TKIs, and patients with both variant 3 and the TP53 mutation had a poor prognosis [77,78]. The efficacy of the combined use of a proteasome inhibitor with alectinib in ALK-rearranged NSCLC cells with TP53 mutation has been shown in vitro and is expected to be applied to clinical practice [79].

Mechanism of Resistance against ALK-TKI
The prognosis of ALK-rearranged advanced NSCLC has improved with the contribution of various therapeutic agents; however, cancer cells develop resistance, and patients eventually progress. There are two types of resistance to ALK-TKI-targeted therapy: primary resistance and acquired resistance.

Acquired Resistance Mechanisms
Mechanisms of acquired resistance are classified into ALK-dependent resistance and ALK-independent resistance.

ALK-Dependent Resistance Mechanisms
ALK-dependent resistance mechanisms include ALK amplification and ALK mutation. ALK amplification has been reported as a resistance mechanism to crizotinib, but its incidence is low, and ALK mutation is the problem in most cases [76]. G1202R mutation is the most common ALK resistance mutation to second-generation ALK-TKIs, but the frequency of ALK resistance mutations depends on the ALK-TKI as prior therapy. A study reported the frequency of ALK mutations by performing biopsies after developing resistance to ALK-TKIs [76]. The common secondary ALK mutations were G1202R (21%), F1174 C/L (17%), and C1156Y (8%) after ceritinib; G1202R (29%), I1171T/S (12%), V11180L (6%), and L1196M (6%) after alectinib; and G1202R (43%), E1210K (29%), D1203N (14%), and S1206Y/C (14%) after brigatinib, respectively. Furthermore, this study presented in vitro IC50 values for ALK-TKIs regarding the different mutations. For instance, IC50 of alectinib for I1171T/S is >50/<200 nM, while the IC50 of ceritinib and brigatinib is reported to be <50. Therefore, subsequent treatment with other ALK-TKIs may be effective depending on ALK resistance mutations. The third-generation ALK-TKI, lorlatinib, has the broadest spectrum for single ALK resistance mutation, including the G1202R mutation. The efficacy of lorlatinib after treatment with a second-generation ALK-TKI was analyzed in 198 patients enrolled in the phase II study, and the prognosis was indicated to be different depending on whether the ALK mutation was the mechanism of resistance to previous therapy. ORR and PFS were 69% and 11.0 months, respectively, in the cohort in which ALK mutations were detected by tissue genotyping, while ORR and mPFS were 27% and 5.4 months in the ALK mutation-negative cohort [86]. The compound ALK mutation (for instance, G1269A + I1171S/C1156, G1202R + L1196M/F1174L, and L1196M + D1203N), which is the main cause of lorlatinib resistance, is the most clinically important major unmet need [87]. The fourth-generation ALK-TKIs are currently considered the most promising treatment for compound ALK mutation. Two fourth-generation ALK TKIs (TPX-0131 and NVL-655) are under development [88][89][90][91]. Both TPX-0131 and NVL-655 can inhibit acquired compound ALK mutations in addition to a wide spectrum of single ALK mutations. A phase I/II clinical trial of TPX-0131 for previously treated ALK-rearranged NSCLC patients (n = 210) is currently ongoing (NCT:04849273).

ALK-Independent Resistance Mechanisms
ALK-independent resistance mechanisms include the activation of bypass signaling pathways, overexpression of p-glycoprotein (p-gp) [92], histological transformation [93], and epithelial-mesenchymal transition (EMT) [94]. The activation of bypass signaling pathways includes EGFR signaling [95,96], amplification of KIT [97], the IGF-1R-IRS-1 pathway [98], MAPK [99], MET amplification [100,101], BRAF V600E mutation [100], activation of the transcriptional co-regulator YAP [102], and HER2-amplification [103]. These can occur during treatment with any ALK-TKI, including lorlatinib, and can lead to resistance to ALK-TKIs. Combination therapy with ALK-TKI and their target drugs in patients with activation of bypass signaling pathways other than ALK may overcome drug resistance; thus, their detection is important [87,104,105]. MET amplification is sensitive to crizotinib. The efficacy of combination therapy with alectinib and crizotinib has been reported in patients in whom MET amplification was detected after progression with alectinib [106].
EMT is the morphological change in which cell-to-cell contacts are lost, making them more mobile and invasive, and tumor cells acquire a mesenchymal morphology and develop drug resistance. HDAC inhibitors have been reported to overcome this resistance mechanism by reversing EMT in vivo and in vitro [94].

Treatment Algorithm for ALK-Rearranged Advanced NSCLC
To date, five ALK-TKIs have been approved by the FDA. However, there are no definitive opinions or the results of clinical trials on sequencing; thus it is largely a matter of clinician judgment today. According to NCCN guideline 2022, alectinib, brigatinib, and lorlatinib are the preferable first-line ALK-TKIs. Ceritinib has not been directly compared with crizotinib in clinical trials, and gastrointestinal toxicity is an issue, although dosage and dose have been adjusted. Crizotinib has been shown to be inferior to other next-generation ALK-TKIs in systemic and central nervous system effects. When the disease progresses during first-line therapy, the identification of resistance mechanisms by performing tissue/liquid biopsy may help to guide optimal treatment. For example, in the case that EML4-ALK G1202R is the cause of resistance, lorlatinib may be a favorable subsequent therapy, and the fourth-generation ALK-TKIs to be developed in the future are effective for compound ALK mutation. When an EGFR mutation is identified, combination therapy with ALK-TKI and EGFR-TKI may be effective, and crizotinib is a reasonable option for patients with confirmed MET amplification. If the disease has converted to small cell lung cancer, a different chemotherapy regimen is required from those for NSCLC. Figure 2 shows the proposed treatment algorithm for ALK-rearranged advanced NSCLC. cell lung cancer, a different chemotherapy regimen is required from those for NSCLC. Figure 2 shows the proposed treatment algorithm for ALK-rearranged advanced NSCLC.

Conclusions
We described the efficacy and safety of six ALK-TKIs, other chemotherapies, and resistance mechanisms. Currently, clinicians have four options as TKIs for first-line treatment: alectinib, ceritinib, lorlatiniba, and brigatinib. In addition, ensartinib is expected to be approved by the FDA in the future. However, there have been no clinical trials directly comparing second-and third-generation ALK-TKIs, of which TKI is the best, which is an issue that needs to be addressed. Furthermore, there are no definite conclusions regarding the treatment sequence after first-line therapy, and further investigation is required. Combination therapy of ALK-TKIs and angiogenesis inhibitors may become an important treatment regimen, as combination therapy of EGFR-TKIs and angiogenesis inhibitors has shown efficacy for a part of EGFR-mutant NSCLC patients. Furthermore, an important matter for angiogenesis inhibitors is that their efficacy may be enhanced when used in combination with IO. The results of clinical trials have shown that IO alone or in combination with IO and platinum doublet chemotherapy is poorly effective, and the combination of TKI and IO was too toxic to continue. The clinical trials of IO in combination with platinum doublet and angiogenesis inhibitors for advanced EGFR/ALK-positive NSCLC

Conclusions
We described the efficacy and safety of six ALK-TKIs, other chemotherapies, and resistance mechanisms. Currently, clinicians have four options as TKIs for first-line treatment: alectinib, ceritinib, lorlatiniba, and brigatinib. In addition, ensartinib is expected to be approved by the FDA in the future. However, there have been no clinical trials directly comparing second-and third-generation ALK-TKIs, of which TKI is the best, which is an issue that needs to be addressed. Furthermore, there are no definite conclusions regarding the treatment sequence after first-line therapy, and further investigation is required. Combination therapy of ALK-TKIs and angiogenesis inhibitors may become an important treatment regimen, as combination therapy of EGFR-TKIs and angiogenesis inhibitors has shown efficacy for a part of EGFR-mutant NSCLC patients. Furthermore, an important matter for angiogenesis inhibitors is that their efficacy may be enhanced when used in combination with IO. The results of clinical trials have shown that IO alone or in combination with IO and platinum doublet chemotherapy is poorly effective, and the combination of TKI and IO was too toxic to continue. The clinical trials of IO in combination with platinum doublet and angiogenesis inhibitors for advanced EGFR/ALK-positive NSCLC are ongoing, and the results are awaited. Resistance against ALK-TKIs is an important issue. Clinical trials of fourth-generation TKI capable of overcoming compound ALK mutation, which is the major mechanism of resistance to lorlatinib, are currently ongoing and are expected to eventually be approved for a treatment option. Moreover, the approach to the ALK-independent resistance mechanism is also important. Especially for patients with activation of bypass signaling pathways, the efficacy of anticancer agents targeting specific genetic mutations in combination with ALK-TKIs has been reported. However, activation of the bypass signaling pathway is rarely identified when the disease progresses in clinical practice, in part because tissue rebiopsy is not often performed. Liquid biopsy is one of the less-invasive methods to identify resistance mechanisms, and non-inferior results compared to tissue samples have been reported. We expect that advances in anticancer therapy and tools for identifying resistance mechanisms, including liquid biopsy, will improve the prognosis of ALK-rearranged NSCLC.
Author Contributions: Writing-original draft preparation, T.F.; writing-review and editing, M.T.; supervision, T.N. and K.K. All authors have read and agreed to the published version of the manuscript.

Conflicts of Interest:
The authors declare no conflict of interest.