The mechanisms of resistance to second- and third-generation ALK inhibitors and strategies to overcome such resistance

Naoki Haratake, Gouji Toyokawa, Takashi Seto, Tetsuzo Tagawa, Tasuro Okamoto, Koji Yamazaki, Sadanori Takeo & Masaki Mori

To cite this article: Naoki Haratake, Gouji Toyokawa, Takashi Seto, Tetsuzo Tagawa, Tasuro Okamoto, Koji Yamazaki, Sadanori Takeo & Masaki Mori (2021): The mechanisms of resistance to second- and third-generation ALK inhibitors and strategies to overcome such resistance, Expert Review of Anticancer Therapy, DOI: 10.1080/14737140.2021.1940964
To link to this article: https://doi.org/10.1080/14737140.2021.1940964

Published online: 21 Jun 2021.

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The mechanisms of resistance to second- and third-generation ALK inhibitors and strategies to overcome such resistance
Naoki Haratakea.,b., Gouji Toyokawac., Takashi Setoa., Tetsuzo Tagawaa., Tasuro Okamotoa., Koji Yamazakic., Sadanori Takeoc. and Masaki Morib.
a.Department of Thoracic Oncology, National Hospital Organization, Kyushu Cancer Center, Fukuoka, Japan; b.Department of Surgery and Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan; c.Department of Thoracic Surgery, Clinical Research Institute, National Hospital Organization, Kyushu Medical Center, Fukuoka, Japan

Received 3 January 2021
Accepted 26 May 2021
resistance; alectinib; ceritinib; brigatinib; lorlatinib

1. Introduction
Lung cancer is the leading cause of cancer-related death worldwide. The prognosis of non-small-cell lung cancer (NSCLC) has been improved with personalized medicine based on the molecular characteristics of patients’ tumors.
A driver oncogene EML4-ALK fusion gene, in which the echinoderm microtubule-associated protein-like 4 (EML4) gene is fused to the anaplastic lymphoma kinase (ALK) gene, was identified in NSCLC in 2007 [1]. Oncogenic ALK gene rearrangements are found in 3–5% of NSCLC patients [1,2]. Until recently, 92 partners that fuse with ALK had been reported [3], and fusion with any of these partner proteins mediates oligomerization of ALK, leading to the constitutive activation of the tyrosine kinase, and aberrant downstream signaling [4–6]. As a result, ALK fusions func- tion as a potent driver oncogene. With the exception of rare cases, the presence of ALK gene rearrangement is mutually exclusive to other driver mutations, such as epi- dermal growth factor receptor (EGFR), ROS1 proto-oncogene receptor tyrosine-protein kinase (ROS1), rearranged during transfection (RET), v-raf murine sarcoma viral oncogene homolog B1 (BRAF), and Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations [5,7,8].
The inhibition of ALK subsequently blocks cell signaling pathways and induces apoptosis of tumor cells. ALK-tyrosine kinase inhibitor (TKI) has shown remarkable efficacy and has

improved the prognosis of patients with ALK-rearranged NSCLC. Crizotinib, a first-generation ALK inhibitor, showed superiority to standard chemotherapy in patients with ALK- rearranged advanced NSCLC [6,8–10], and until recently, crizo- tinib was the standard first-line treatment for ALK-rearranged NSCLC [6]; however, most patients treated with crizotinib relapse within about one to several years via both ALK- dependent (i.e. on-target resistance) and ALK-independent mechanisms (i.e. off-target resistance) [5,7,11–13].
To overcome this resistance to crizotinib, some structurally distinct, next-generation ALK-TKIs (second-generation ALK- TKIs: alectinib, ceritinib, and brigatinib; third-generation ALK- TKI: lorlatinib) have been developed [7,13–34]. In general, these drugs are more potent than crizotinib and are effective against ALK-dependent mechanisms, such as some known secondary mutations [5,7,27,35–39]. Because of their good efficacy in the most of crizotinib-resistant patients, these next- generation ALK-TKIs have become standard treatments [15– 17,22,23,26,27,40–42].
In addition, the superiority of second-generation ALK-TKIs to crizotinib has recently been demonstrated during the first- line treatment in several phase III trials [18,29,30,43]. Alectinib is the first ALK-TKI to show superiority to crizotinib in a first- line setting in two randomized phase III trials [29,30]. Brigatinib also has significantly prolonged progression-free survival (PFS) in comparison to crizotinib [18]. Thus, second-

CONTACT Naoki Haratake [email protected] Department of Thoracic Oncology, National Hospital Organization, Kyushu Cancer Center, Fukuoka, Japan
© 2021 Informa UK Limited, trading as Taylor & Francis Group

generation ALK-TKIs have become the standard treatment for treatment-naïve patients with ALK-positive NSCLC; however, as with crizotinib, patients develop acquired resistance, with ALK- dependent resistance accounting for approximately 50% of cases of acquired resistance, and with ALK-independent resis- tance accounting for the remaining 50% [5,13].
Although several studies on resistance to crizotinib have been reported [39,40,44,45], the mechanisms of resistance to second- and third-generation ALK-TKIs remain to be fully elucidated. To improve the prognosis of patients with ALK- rearranged NSCLC, it is necessary to comprehensively clarify these mechanisms of resistance.
We herein review the mechanisms of resistance to next- generation ALK-TKIs that have been identified in both clinical and pre-clinical settings, and introduce strategies for overcom- ing resistance and discuss ongoing clinical trials.

2. Resistance mechanisms
2.1. ALK-dependent resistance (On-target resistance)
ALK-dependent resistance, which is observed in 30% of crizo- tinib-resistant and 50% of second-/third-generation ALK-TKI- resistant cases, is mediated by a genetic alteration in ALK itself. ALK-dependent resistance includes either a second-site muta- tion (secondary mutation) in the tyrosine kinase domain that interferes with ALK-TKI binding or the amplification or loss of the ALK fusion gene [5,7,13,39]. To date, several secondary mutations in ALK have been reported to mediate resistance to ALK-TKI [5,7,9,11–13,21,36–38,46–51]. The list of mutations associated with ALK-dependent resistance is shown in Figure 1 and Table 1.

2.1.1. Gatekeeper mutations
A secondary mutation, L1196M, has been reported to be an analogous gatekeeper mutation that leads to ALK-TKI resis- tance [52]. While there are more types of secondary mutations showing resistance to ALK-TKIs than to EGFR-TKI, the L1196M gatekeeper mutation is predominant, occurring in 7% of cri- zotinib-resistant cases. This mutation sterically inhibits ALK-TKI binding [5].

2.1.2. Solvent-front mutations
Solvent-front mutations, which occur in the solvent-exposed region of the ALK kinase domain, close to the binding site of first- and second- ALK-TKIs, limit the binding of ALK-TKIs via steric hindrance due to the presence of a large basic residue [5,53,54]. G1202R, D1203N, and S1206 mutations have been reported as solvent-front mutations showing resistance to

Figure 1. This heatmap shows the sensitivity of each ALK-TKI based on in vitro data [5,13,103] the database of the Catalogue of Somatic Mutations in Cancer (COSMIC), and clinical data [11,13,15,16,19,21,24,25,33,36–40,47–50,69,74,91].
Green: all studies reported sensitivity to the ALK TKI and showed C50 < 50 nmol/L, or sensitivity to each ALK-TKI in the clinical setting. Green yellow: one study showed C50 < 50 nmol/L, but another study showed 50 nmol/L < IC50 < 200 nmol/L.
Yellow: all studies reported sensitivity to the ALK-TKI and showed 50 nmol/L< IC50 < 200 nmol/L, or showed both sensitivity and resistance to each ALK-TKI in the clinical setting. Orange: one study showed 50 nmol/L < IC50 < 200 nmol/L, but another study showed 200 nmol/L< IC50.
Red: all studies reported sensitivity to the ALK-TKI and showed 200 nmol/L < IC50, or resistance to each ALK-TKI in the clinical setting.

Table 1. The resistance mechanisms in patients with ALK-positive NSCLC.

2.1.4. Compound mutations
In addition to the single mutations mentioned above,

ALK-dependent resistance


diverse ALK kinase domain compound mutations were iden-

ALK-TKI (On-target resistance) (Off-target resistance)

tified, mainly in lorlatinib-resistant cells or patient samples

Alectinib L1151tins F1171N V1180L G1202del G1202R G1206C G1206Y E1210K F1245C G1269A G1269S
Compound mutations

Ceritinib G1123S T1151K T1151 R L1151tins
L1152P L1152R C1156Y F1174C F1174L F1174V L1198F G1202del G1202R G1206C G1206Y E1210K F1245C
Compound mutations

Brigatinib G1202R D1203N G1206C
G1202R-containing compound mutations
T1151 R+ C1156Y+E1161D

Lorlatinib G1202R-containing compound
mutations C1156Y+L1198F I1171S + G1269A

cMET activation Coactivation of MET and SRC
The overexpression of ABCC11
Transformation to SCLC EMT
MEK mutation Overexpression of
p-glycoprotein ABCB1, SHP2
Activation of SRC, EGFR, KIT or IGF-1 R
MTOR T1834-T1837del,
JAK3R948C mutation CDKN2A/B loss-of- function mutation[ NFE2L2E79Q mutation MET amplification BRAFV600E and KRASG12D
mutations Activation of EGFR
TP53, NRASG12D, and
MAP3K1 mutations NF2 loss-of-function mutations
Transformation to neuroendocrine carcinoma

after these patients received sequential ALK-TKI treatments [13]. The previously reported compound mutations are shown in Figure 1. Although most compound mutations are associated with resistance to all ALK-TKIs, including lorlatinib, a few compound mutations that were re- sensitized to first- or second-generation ALK inhibitors (e.g. C1156Y + L1198F became re-sensitized to crizotinib, and I1171N + L1256F became re-sensitized to alectinib; Figure 1)[13,24].

2.2. ALK-independent mechanisms (Off-target resistance)
Alterations of genes other than the ALK fusion gene in tumor cells also cause ALK-TKI resistance. To date, several ALK- independent mechanisms of crizotinib have been reported [20,36,40,44,47,53,55,57]. The list of mutations associated with ALK-independent resistance is shown in Figure 2 and Table 1.

2.2.1. Activation of downstream pathways
The activation of downstream signaling pathways can avoid reliance on upstream signaling from the ALK fusion gene. MAPK pathway activation was shown to have a significant

ALK, anaplastic lymphoma kinase; NSCLC, non-small cell lung cancer; SRC, Proto- oncogene tyrosine-protein kinase Src; YAP 1, Yes-associated protein 1; ATP- binding cassette subfamily C member 11, ABCC11; SCLC, small cell lung cancer; EMT, Epithelial-to-mesenchymal transition; SHP2, SRC homology 2 domain; EGFR, epidermal growth factor receptor; KIT, v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog; IGF1-1 R, insulin-like growth factor 1 receptor; MTOR, mammalian target of rapamycin; JAK3, Janus Kinase 3; BRAF, v-raf murine sarcoma viral oncogene homolog B1; KRAS, Kirsten rat sarcoma viral oncogene; NF2, homolog; Neurofibromatosis type 2

crizotinib [5,13,53,54]. G1202R, which is the most frequent resistance mutations against second-generation ALK-TKIs, mediates resistance to all first- and second-generation ALK- TKIs [5,13,53,54].

2.1.3. Other second-site mutations
Other mutations are present in functionally important residues of the ALK kinase domain, altering the ATP affinity or inhibit- ing the ALK-TKI binding and inducing resistance to ALK-TKIs. In ALK-rearranged NSCLC, there are more types of secondary mutations that confer resistance to crizotinib in comparison- to second- or third-generation ALK-TKIs. G1269A mutation, which is found in the ATP binding pocket, sterically inhibits ALT-TKI binding [55]. ALK mutations near the αC helix (1151Tins, L1152R, C1156Y, and F1174C) induce resistance by changing its conformation, which changes the kinase activity without limiting the ALK-TKI binding directly [39,52,53,56].
Additional resistance mutations are mentioned in detail in the sections on each ALK-TKI.

effect on ALK-TKI resistance. This is described in detail in the ceritinib section. In addition, an activating mitogen-activated protein kinase 1 (MEK 1) mutation is associated with the crizo- tinib resistance and has sensitivity to a MEK inhibitor [58]. Phase I and II studies are ongoing to evaluate the combined use of ALK and MEK inhibitors (Table 1).
Interestingly, it has never been reported that JAK–STAT3 or PI3K–AKT pathway gene mutations caused resistance to crizo- tinib; a further analysis is therefore necessary to clarify the off- target mechanism of ALK-TKI resistance.

2.2.2. Activation of parallel pathways
Some activation of parallel signaling pathways via other receptor tyrosine kinases leads to the proliferation and sur- vival of tumor cells [59]. The activation of parallel pathways via the activation of EGFR-, KRAS-, v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT)-, and insulin-like growth factor 1 receptor (IGF-1 R)-mediated signaling path- ways, induces resistance against crizotinib [5,53,55,57,59– 61]. In addition, it has been reported that HER2 and HER3 activations are also associated with crizotinib resistance [60]. On the other hand, MET amplification, which was found in 5%-20% of EGFR-TKI-resistant cases, is not associated with crizotinib resistance because crizotinib is a MET inhibi- tor [46].

2.2.3. Histological transformation
The histological transformation to a small-cell lung cancer (SCLC) has been reported as EGFR- or ALK-TKI resistance mechanisms in a subset of treated NSCLC patients [62,63].

Figure 2. Signaling pathways in ALK-independent resistance mechanisms.
Anaplastic lymphoma kinase (ALK)-independent resistance mechanisms include activation of parallel pathways (I), activation of downstream pathways (II), and histological transformation (III). cMET activation, over expression of ATP-binding cassette subfamily C member 11 (ABCC11), p-glycoprotein ABCB1, activation of epidermal growth factor receptor (EGFR), v-kit Hardy- Zuckerman 4 feline sarcoma viral oncogene homolog (KIT), and insulin-like growth factor 1 receptor (IGF-1 R) were identified as ALK-independent resistance mechanisms with activation of downstream pathways. In addition, over expression of SRC homology 2 domain (SHP2), mutation of v-raf murine sarcoma viral oncogene homolog B1 V600E (BRAF V600E), Kirsten rat sarcoma viral oncogene homolog (KRAS G12D), MEK, mechanistic target of rapamycin (mTOR), and Janus kinase 3 (JAK3), and activation of SRC were identified as ALK-independent resistance mechanisms with activation of downstream pathways. With regard to histological transformations which mediate second- and third-generation ALK-TKIs resistance, the cases with transforming to epithelial-to-mesenchymal transition (EMT), small cell carcinoma, and neuroendocrine carcinoma were reported.
Circle A, the resistance mechanism to alectinib; Circle C, the resistance mechanism to ceritinib; Circle B, the resistance mechanism to brigatinib; Circle L, the resistance mechanism to lorlatinib.
ALK, Anaplastic lymphoma kinase; TKI, tyrosine kinase inhibitor; ABCC11, ATP-binding cassette subfamily C member 1; EGFR, epidermal growth factor receptor; KIT, v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog; IGF-1 R, insulin-like growth factor 1 receptor; SRC homology 2 domain, SHP2; BRAF, v-raf murine sarcoma viral oncogene homolog B1; KRAS, Kirsten rat sarcoma viral oncogene homolog; mTOR, mechanistic target of rapamycin; JAK3, Janus kinase 3; YAP1, yes-associated protein 1 (YAP 1); EMT, Epithelial-to-mesenchymal transition.

Furthermore, in a case report, the transformation to sarcoma- toid carcinoma showed crizotinib resistance in an ALK-positive adenocarcinoma patient [64].
The epithelial-to-mesenchymal transition (EMT) is another histological transformation found in some cases that are resistant to several anticancer treatments, including EGFR- and ALK-TKIs, which manifests as a series of cellular altera- tions favoring a more invasive, mesenchymal phenotype [65,66]. The EMT is the process through which cells down- regulate their epithelial characteristics and acquire a mesenchymal phenotype. The EMT cause fibrosis, tumor invasion, and cancer progression [65]. Under the EMT, the junction of epithelial cells is lost, their cytoskeletons are reor- ganized, and their gene expression is reprogrammed to acquire motility and an invasive phenotype. E-cadherin is an epithelial marker that maintains cell–cell adhesion and inhi- bits cell mobility and invasiveness. Vimentin is a type III intermediate filament that is a marker of the mesenchymal phenotype of the EMT. The downregulation of the expression of E-cadherin and the concomitant upregulation of the expression of vimentin are common indicators of the EMT in carcinomas [66].
Some of the abovementioned types of resistance (I–III) have been reported to mediate resistance to second- and third-generation ALK-TKIs. The detailed mechanisms are mentioned in the sections for individual ALK-TKIs.

3. Second-generation ALK inhibitors
3.1. Alectinib
Alectinib selectively and potently blocks two receptor tyrosine kinase enzymes: ALK and RET proto-oncogene [67]. The IC50 of ALK (2 nmol/L) is much lower than that of crizotinib [29,30]. Alectinib is the first ALK-TKI that was compared to crizotinib in a first-line setting in two randomized studies: the J-ALEX trial, which was a phase III trial performed in Japan, and the ALEX trial, which was an international phase III trial [29,30].
In the ALEX trial, all enrolled patients received alectinib in a first-line setting. The median investigator-assessed PFS was significantly longer with alectinib than with crizotinib (34.8 months vs. 10.9 months; hazard ratio [HR], 0.43; 95% con- fidence interval [CI], 0.32–0.58; P < 0.001). Because crossover was not permitted in the protocol treatment of the ALEX trial, it is difficult to compare the strategies of upfront and sequential use of alectinib. Recently, the final, mature PFS data, together with the OS were reported from ALEX (data cutoff:
30 November 2018). The median OS was not reached with alectinib and was 57.4 months with crizotinib (HR 0.67, 95% CI 0.46–0.98). Although formal statistical testing of OS was not planned, a clinically meaningful difference was observed in the 5-year OS rate (alectinib, 62.5%; crizotinib, 45.5%) [68]. In addi- tion, alectinib was shown to be more effective than crizotinib for preventing the development of brain metastasis [34].

With regard to crizotinib-pretreated cases, alectinib had significantly greater efficacy than chemotherapy in the phase III ALUR study (PFS, 9.6 months vs. 1.4 months; HR, 0.32; 95% CI, 0.17–0.59) [16]. Thus, alectinib has also become a standard treatment for crizotinib-refractory patients.

3.1.1. ALK-dependent resistance (On-target resistance)
As described above, alectinib has shown great efficacy among crizotinib-refractory cases, partly because alectinib has sensi- tivity to some secondary mutations, such as L1196M or G1269A mutations, which mediate crizotinib resistance [16,31,37,51]. With regard to the ceritinib-refractory cases, alectinib was shown to be active against ceritinib-resistant cases with G1123S mutations [21].
However, the resistance mutations are more frequently found in cases treated with alectinib than in those treated with crizotinib. Gainor et al. reported that ALK resistance mutations were found in 53% of biopsy specimens of alecti- nib-resistant tumors [5]. It was reported that G1202R, V1180L, and I1171T/N/S mutations were associated with resistance to alectinib [5,37,53,55,56,69]. Of those, the I1171T mutation is the second-most common resistance mutation showing resis- tance against alectinib. It distorts the αC helix and changes the residue position, thereby inhibiting binding of alectinib [5,37]. G1202R, which was found in 29% of alectinib cases in the report by Gainor et al., is the most common mutation to mediate resistance to second-generation ALK-TKIs, including alectinib [5,13,51].
With regard to other ALK resistance mutations, G1202del, G1206C, G1206Y, E1210K, F1245C, G1269A, and G1269S muta-
tions have been found to be alectinib-resistant with an in vitro
IC50 of >200 nM (Figure 1) [5,9,13,37,49,69,70].

3.1.2. ALK-independent mechanisms (Off-target resistance) C-MET activation. c-MET, which is a receptor tyro- sine kinase, is expressed on the surfaces of various cells [46]. Hepatocyte growth factor (HGF) is the ligand of that receptor, and in cancer cells, its binding to c-MET promotes tumor development by activating Ras/MAPK and PI3K-AKT-mTOR signaling [46,71].
Some reports have shown that c-MET activation, which is closely related to c-MET overexpression, amplification, and gene expression, mediates acquired resistance against alecti- nib [46,71–73]. Although there is no clear evidence of the effectiveness of MET inhibitors against alectinib refractory cases in a clinical setting, theoretically, MET inhibitors like crizotinib can overcome resistance mediated by c-Met activa- tion [46,72]. In a previous case report, crizotinib showed good efficacy against an alectinib-resistant case with MET gene amplification [46]. Coactivation of MET and Proto-oncogene tyrosine-protein kinase Src (SRC). SRC is an intracellular tyrosine kinase involved in the differentiation and survival of tumor cells. SRC activates several downstream receptor tyro- sine kinases [74]. Tsuji et al. compared and analyzed patient- derived paired cell lines before treatment and after the devel- opment of alectinib resistance and identified the co-activation

of c-SRC and MET in the cell lines with alectinib resistance [75]. The SRC and MET salvage signaling pathways were associated with alectinib resistance, anti-apoptosis, and tumor growth. In vitro, it was shown that the combined use of inhibitors against SRC and MET significantly restored alectinib sensitivity. In addition, in vivo, it was shown that triple inhibition of SRC, ALK, and MET overcame alectinib resistance [75]. Yes-associated protein 1 (YAP 1). YAP1 is a transcriptional regulator with oncogenic activity that is involved in tumor genesis, cellular proliferation, and mainte- nance of stem cells serving as a Hippo pathway effector through its binding with transcription factors that promote cell proliferation and inhibit apoptosis [76]. Tsuji et al. used a proteome approach with patient-derived cells and evaluated the efficacy of alectinib against ALK-rearranged cells. They found that activated YAP1 plays an important role in the survival of ALK-positive cells after alectinib treatment by reg- ulating apoptosis [77]. YAP1 mediates alectinib-resistance via the anti-apoptotic factors Mcl-1 and Bcl-xL, and inhibiting YAP1 and ALK was associated with a longer tumor remission than alectinib monotherapy in vivo [77]. Therefore, combining the use of a YAP1 inhibitor with alectinib might be effective in alectinib-refractory cases. Amphiregulin. Patients who develop leptomenin- geal carcinomatosis, which occurs frequently in ALK-positive NSCLC, often show resistance to ALK-TKIs, including alectinib. Arai et al. successfully cultured an alectinib-resistant cell line from a leptomeningeal carcinomatosis mouse model [78]. An analysis of the alectinib-resistant cell line showed that amphir- egulin-triggered EGFR activation mediate the resistance to alectinib in the leptomeningeal space. Amphiregulin levels of the cerebrospinal fluid were found to be high in alectinib- refractory NSCLC patients with leptomeningeal carcinomatosis [78]. The combined use of alectinib and osimertinib could reportedly overcome that resistance in the leptomeningeal carcinomatosis model mouse. The overexpression of ATP-binding cassette sub- family C member 11 (ABCC11). ABCC11 is a member of the superfamily of ATP-binding cassette (ABC) transporters. Various molecules are transported through the ABC proteins on cellular membranes. ABC genes are divided into seven distinct subfamilies, among which ABCC11 transporter is reported to be associated with multi-drug resistance. Recently, it was reported that the high expression of ABCC11 might mediate resistance to alectinib [79]. In vitro, the expres- sion of ABCC11 was significantly higher in alectinib-resistant cell lines than in an alectinib-sensitive cell line, and the inhibi- tion of ABCC11 overcame resistance to alectinib in vitro [79]. Therefore, in alectinib refractory cases with the overexpression of ABCC11, ABCC11 inhibition combined with alectinib might overcome resistance. Transformation to SCLC. Histological transforma- tion of adenocarcinoma to SCLC has been reported in some crizotinib-refractory cases, and in one alectinib-resistant case [80]. In that case, after a partial response to alectinib was

observed in all targeted lesions, the primary lesion increased in size, whereas other metastatic lesions showed a marked decrease in size. A biopsy of the primary lesion revealed ALK rearrangement and transformation into SCLC [80]. EMT. Although EMT is reported to be associated with several molecular-targeted drugs, little is known about its rela- tionship with ALK-TKI resistance. Recently, it has been shown that the mesenchymal phenotype caused by EMT, which was associated with a low expression of miR-200 c and high expres- sion of Zinc Finger E-Box Binding Homeobox 1 (ZEB1), can coex- ist with secondary mutations as an independent mechanism of resistance against ALK-TKIs. Both in vitro and in vivo, treatment with a histone deacetylase inhibitor showed efficacy against the resistance with EMT by reverting it [66].

3.2. Ceritinib
Ceritinib is a second-generation ALK-TKI that is active not only in conventional ALK-positive tumors but also in those expressing some secondary mutations and the insulin growth factor 1 receptor (IGF1R) [43]. In addition, ceritinib is also anti-ROS TKI [81]. The IC50 of ceritinib to ALK-positive NSCLC is low (150 pM in vitro; 20- to 30-fold lower than that of crizotinib) [81].
Based on the results of the ASCEND-4 study, ceritinib is the first second-generation ALK-TKI approved as a first-line treat- ment for ALK-positive NSCLC. In the ASCEND-4 study, ceritinib significantly prolonged the PFS in comparison to chemother- apy (PFS, 16.6 vs. 8.1 months; HR, 0.55; 95% CI, 0.42–0.73;
p < 0.001) [43].
In the ASCEND-5 phase III trial, the significant clinical ben- efit of ceritinib in comparison to chemotherapy was shown in ALK-positive NSCLC patients after crizotinib failure (PFS, 5.4 vs. 1.6 months; HR, 0 · 49; 95% CI, 0.36–0.67; p < 0.001) [27]. With
regard to the dose of ceritinib, these initial trials were con- ducted using a dose of 750 mg, daily, in fasted patients. The ASCEND 8 trial, which is a randomized open-label Phase 1 study, demonstrated equivalence between this dose and 450 mg, daily, with food, and the lower dose was associated with decreased gastrointestinal toxicity [82]. The approved dose of ceritinib is 450 mg, orally, once daily with food.

3.2.1. ALK-dependent resistance (On-target resistance) Ceritinib also has sensitivity to several secondary mutations that mediate crizotinib resistance, as shown in ASCEND-5 phase III trial [5,13,39].
In in vitro studies using Ba/F3 cells with EML4-ALK, Ba/F3 cells transfected with mutants, such as L1196M, S1206Y, G1269A, and G1269S, were shown to be sensitive to ceritinib [5,13,39]. On the other hand, ceritinib is not active against cells resistant to crizotinib with the 1151Tins, L1152P, C1156Y, F1174C, and G1202R mutations [5,13,39,82].
In the clinical setting, the efficacy of ceritinib in crizotinib- refractory patients with secondary mutations of L1196M, G1269A, 1151Tins, and S1206Y was confirmed [5]. Regarding alectinib resistance mutations, I1171T/N/S and V1180L are sensitive to ceritinib [5,13,39,82].
Gainor et al. reported that 54% of ceritinib-resistant speci- mens were detected with secondary mutations, and 17%

harbored compound mutation (>2 secondary mutations). The most common ALK mutations were G1202R (21%) and F1174C/ L (17%). In another study analyzing 11 biopsied tumors from patients treated with ceritinib after crizotinib, the G1123S, F1174C/L/V, and G1202R mutations mediated resistance to cer- itinib [39]. As mentioned in the alectinib section, among these secondary mutations, the G1202R mutation shows resistance to all second-generation ALK-TKIs [5]. In contrast, the F1174C/L/V mutations, which confer resistance to ceritinib, are sensitive to both alectinib and lorlatinib [5,39,48,83,84]. In addition, a recent case study reported that a patient treated with ceritinib who developed a T1151 R mutation showed a dramatic response with brigatinib [50] (Figure 1).

3.2.2. ALK-independent mechanisms (Off-target resistance) Activating mutation of MEK. Dual-specificity pro- tein phosphatase 6 (DUSP6: a member of the MAPK phospha- tase family) downregulation or amplification of KRAS (wild- type) activates the downstream of MAPK pathway, resulting in resistance to ceritinib [83]. DUSP6 interacts with targeted extracellular signal-regulated kinase 1/2 specifically through negative feedback regulation in the MAPK pathway. In in vitro and in vivo NSCLC models, targeting downstream MAPK signaling with a MEK inhibitor combined with ceritinib has been found to overcome this resistance [83]. The overexpression of p-glycoprotein ABCB1. ABC transporters, including p-glycoprotein ABCB1, transport hydrophobic substrates across the blood-brain barrier or from cells. A previous study analyzing ceritinib-resistant patient-derived cells reported that the overexpression of p-glycoprotein ABCB1 induced resistance to crizotinib and ceritinib [84]. The resistance was shown to be overcome with alectinib or lorlatinib. In the clinical setting, it was reported that the overexpression of p-glycoprotein was found in 3 of 13 patients who had been treated with ceritinib or crizotinib; however, the clinical efficacy of p-gly- coprotein inhibitors is still unclear [84]. SRC activation. The SRC pathway has been reported to be involved in the mechanism underlying resis- tance to ceritinib, as shown using patient-derived cell culture models [85]. Knockdown of SRC alone with siRNA effectively sensitized ceritinib resistance in ALK-positive cells, and SRC inhibition by AZD0530 was effective in ALK-resistant cancer cells [85]. Overexpression of SRC homology 2 domain (SHP2). SHP2, a nonreceptor protein tyrosine phosphatase, was identified as a common targetable resistance node using a shRNA screen of 1,000 genes in multiple-patient-derived cells that showed resistance to ceritinib [86]. SHP2 activates multiple downstream tyrosine kinases that mediate resistance to ceritinib. The combined use of SHP2 and ceritinib inhibited the growth of resistant patient-derived cells by preventing compensatory RAS and ERK1/2 reactivation [86]. These find- ings suggest that combined ALK and SHP2 inhibition might be effective for treating ceritinib-refractory NSCLC. Activation of IGF-1 R, KIT, and EGFR. Activation of IGF-1 R, KIT, and EGFR was reported to be associated with resistance to ceritinib, just as with crizotinib resistance. In an in vitro study using an NSCLC cell line (H3122) with the EML4- ALK variant 1, the overexpression of receptor tyrosine kinases (p-IGF-1 R, p-HER3, or p-EGFR) was observed in cell lines with ceritinib resistance [48]. In addition, amphiregulin, neuregulin- 1 (NRG1), a ligand for human epidermal growth factor recep- tor 3 (HER3), was shown to be upregulated and to be respon- sible for ceritinib and alectinib resistance by activating the EGFR family pathways through the NRG1-HER3-EGFR axis [87]. It was revealed that combination treatment with afatinib was effective to overcome this resistance. As mentioned above, it was reported that ceritinib inhibited the phosphor- ylation of both ALK and IGF-1 in in vitro experiments; thus, IGF- 1R activation-mediated resistance might not be observed in ceritinib-refractory case [38,83]. EMT. It was reported that among 11 ceritinib- resistant re-biopsy specimens, 5 (42%) specimens showed findings of EMT with the diffuse expression of vimentin and the loss of E-cadherin staining. Secondary mutations, includ- ing two cases with ALK L1196M, were found in three of these five specimens; however, since we observed some concomitant alterations that might also contribute to resis- tance, EMT might not be the only cause of resistance in these cases [5]. Further pre-clinical studies are necessary to reveal the mechanisms underlying resistance in association with EMT.

3.3. Brigatinib
Brigatinib is a highly potent and selective ALK and ROS1 inhibitor [38]. Brigatinib potently inhibited the in vitro kinase activity of ALK (IC50, 0.6 nmol/L) and five secondary mutations (C1156Y, F1174L L1196M, R1275Q, and G1202R) [38].
Brigatinib showed good efficacy in ALK-positive NSCLC patients pretreated with crizotinib in phase II clinical trial [18]. Because of severe adverse events observed in the phase I/II clinical trial, in the phase II study, among 222 patients who received one of the two brigatinib dosing regimens (90 mg, once daily vs. 180 mg, once daily with a 7-day lead-in at 90 mg), the ORR was 45% and 54%, and median PFS was
9.2 months (95% CI, 7.4 to 15.6) and 16.7 months (95% CI,
11.1 to not reached), respectively [20].
With regard to initial treatment, in a phase III trial that included 275 patients, brigatinib at a dose of 180 mg, once daily (with a 7-day lead-in period at 90 mg) showed super- ior efficacy to crizotinib (estimated 12-month PFS, 67% for brigatinib vs. 43% for crizotinib; p < 0.01) [18]. The overall survival data from these phase 3 trials are currently imma- ture. In preliminary reports, the median PFS to an indepen- dent review committee was 24 vs. 11 months, respectively (HR 0.49, 95% CI 0.35–0.68) [88]. Among patients with baseline brain metastasis, even greater PFS benefits were observed. Among those with brain metastasis at baseline,

the intracranial response rate was significantly higher with brigatinib (78% vs. 26%) [18].

3.3.1. ALK-dependent resistance (On-target resistance) Brigatinib has been reported to be sensitive to almost all crizotinib-, alectinib-, and ceritinib-resistant secondary muta- tions, including the G1202R mutation in vitro [38]. On the other hand, G1202R mutation was identified in the brigatinib- resistant cases [5,13,19]. Gainor et al. reported six brigatinib- refractory cases that were analyzed with next-generation sequencing (NGS). G1202R was found in three cases, two compound mutations (E1210K+S1206C and E1210K+D1203N) were simultaneously found in three cases, and no mutation was detected (wild-type) in two cases [5]. Thus, G1202R might mediate resistance to brigatinib.
In the analysis of plasma specimens from the initiation of brigatinib treatment and the end of brigatinib treatment in crizotinib-resistant ALK-positive NSCLC patients enrolled in phase I/II or pivotal phase II trials, 67 plasma samples were evaluated [40]. ALK fusion was detected in 45% of these plasma samples [40]. Among the ALK-positive patients, 33% had secondary ALK mutations (ORR: 50%) and 67% did not (ORR: 60%). The best responses in patients with secondary ALK mutations were as follows: a complete response in two patients (ALK amplification copy number = 10; T1151M); a par- tial response in three patients (L1196M; E1408V; amplification copy number = 6); stable disease in four patients (L1196M; E1419K; F1174C; C1156Y+S1206F+G1269A); and progressive
disease in one patient (T1151R+C1156Y+E1161D+F1174L) [40]. Based on this analysis, brigatinib seems to have sensitiv- ity to ALK amplification, T1151M, L1196M, and E1408V muta- tions, and compound mutations mediate resistance to brigatinib as expected.
In a multicenter retrospective study of 22 patients treated with alectinib followed by brigatinib, the ORR of brigatinib was 17%, and the median PFS was 4.4 months (95% CI: 1.8– 5.6) [19]. Nine patients underwent a repeat biopsy at the time of progression of alectinib before starting brigatinib. ALK resis- tance mutations were identified in six (67%) cases. The six resistance mutations were I1171T (n = 1), I1171N (n = 2), V1180L (n = 1), and G1202R (n = 1). The case with the V1180L mutation only achieved a partial response, and this case was still receiving brigatinib at the time of data cutoff (PFS was more than 4 months). Three cases (I1171T [n = 1], I1171N [n = 2]) had stable disease, and the one case with a G1202R mutation had PD on the first tumor re-assessment [13].
As mentioned in the ceritinib section, it was reported that a patient treated with ceritinib, who developed a T1151R mutation, showed a great response with brigatinib; however, the PFS was only 3 months [50].
These clinical results suggest that the clinical benefit of brigatinib is still unclear in cases involving secondary muta- tions, especially for the secondary mutations mediated by alectinib. This might be due to the wide variety of resistance mechanisms of alectinib.

3.3.2. ALK-independent mechanisms (Off-target resistance) MTOR T1834-T1837del, JAK3R948C mutation, CDKN2A/B loss, and NFE2L2E79Q mutation. The study using next-generation sequencing by Yoda et al. included two brigatinib refractory cases (case 1: 1st line treatment with brigatinib; case 2: second-line treatment with brigatinib after crizotinib). In case 1, no ALK mutation or off-target mechanism was detected. On the other hand, in case 2, the compound mutation D1203N+E1210K and other genetic alterations (MTOR T1834_T1837del, JAK3R948C, CDKN2A/B loss, and NFE2L2E79Q) were confirmed. This compound mutation or these genetic alterations might be associated with brigatinib- resistance [40]. MET amplification, mutation of BRAF-V600E, and KRAS -G12D. In the above analysis of phase I/II and pivotal phase II (ALTA) trials, 20 plasma specimens were evaluated after brigatinib failure [40]. Complex mutation patterns were reported to be associated with resistance in five specimens (25%): (1) High-level ALK-amplification (copy number = 58),
(2) ALK-amplification (copy number = 14) and MET- amplification (copy number = 6), (3) compound mutation with S1206F+S1206C and ALK-amplification (copy num- ber = 6), (4) compound mutation with G1202R+L1196M
+L1198Q, (5) secondary mutation G1202R, mutation with BRAF-V600E, and mutation with KRAS-G12D. Of these, MET amplification, mutation of BRAF-V600E, and KRAS -G12D might be associated with the off-target resistance mechan- isms of brigatinib. BRAF fusion and BRAF gene mutations were reported to be a mechanism of resistance to EGFR-TKI [89,90]. Further exploration is necessary to clarify these off- target resistance mechanisms of brigatinib in vivo and in vitro.

3.4. Ensartinib
Ensartinib is a potent and dual ALK and MET inhibitor with IC50 values of <0.4 nM and 0.74 nM, respectively. In the preliminary results of the eXalt3 trial, which is phase III randomized study comparing ensartinib to crizotinib in
290 patients with ALK-positive NSCLC, those assigned to receive ensartinib showed an improvement in median PFS relative to those assigned to receive crizotinib (26 months vs. 13 months, respectively; HR, 0.51; 95% CI, 0.35–0.72) [91]. Ensartinib is a relatively new ALK-TKI; thus, few resistance mechanisms have been reported.

4. Third-generation ALK inhibitor
4.1. Lorlatinib
Lorlatinib, which is a reversible ATP-competitive macrocyclic third-generation ALK/ROS1-TKI, has been shown to be active against almost all ALK secondary mutations, including the G1202R mutation [92]. In addition, lorlatinib has the capability to cross the blood–brain barrier at sufficient concentrations to be effective against brain metastases, partly because lorlatinib is not a substrate of p-glycoprotein [17,92,93]. In the phase

I trial, the mean ratio of cerebrospinal fluid to plasma lorlatinib concentration was 0.75, and this result proved the significant CNS penetrability of lorlatinib [17].
In the phase II study of lorlatinib, 276 patients were enrolled in six different expansion cohorts based on prior treatment [23]. Among the 198 patients who received at least one previous ALK-TKI before enrolling in the trial, the ORR was 47.0% (95% CI 39∙9–54∙2). Of the 81 patients with measurable baseline CNS lesions, the intracranial response rate was 63%, and the estimated median intracranial duration of response was 14.5 months (95% CI 8.4–14.5). Among 59 previously treated with only crizotinib, ORR was 70%, and median PFS was not reached (95% CI: 11∙1–NR) [23]. On the other hand, among 28 patients treated with one second- generation ALK-TKI, ORR was 32%, and the median PFS was 5.5 months (95% CI: 2.7–9.0) [23].
Based on the above trials, in 2018, the U.S. Food and Drug Administration approved lorlatinib for patients with ALK- positive metastatic NSCLC whose disease had progressed on crizotinib and at least one other ALK-TKI for metastatic dis- ease; or whose disease had progressed on alectinib or ceritinib as the first ALK-TKI therapy for metastatic disease. In addition, the results of the phase III CROWN trial comparing lorlatinib with crizotinib in the first-line treatment of the patients with ALK-positive stage IIIB/IV NSCLC were recently reported [93]. Among the 296 patients, lorlatinib significantly prolonged PFS in comparison to crizotinib (PFS, NR vs. 9.3 months; HR, 0.28; 95% CI, 0.19–0.41; p < 0.001) at first interim analysis on the trial (approximately 18 months of follow-up). Grade 3–4 adverse events occurred in 72% of patients who received lorlatinib and 56% of patients who received crizotinib [93]. Thus, lorlatinib would be a standard treatment option for treatment-naïve patients with ALK-positive advanced NSCLC; however, as with other 1st- and second-generation ALK-TKIs, acquired resistance to lorlatinib develops in almost all patients [11,13,94].

4.1.1. ALK-dependent resistance (On-target resistance)
As previously described, lorlatinib is active against almost all ALK secondary mutations [5,13]; however, it was reported that compound mutations were found in approximately 35% of lorlatinib-refractory cases [11]. The analysis of repeated tumor biopsies from lorlatinib-refractory patients revealed that compound ALK mutations developed in a stepwise fashion in patients treated with sequential ALK- TKIs [13]. The most common on-target resistance mechan- ism in patients relapsing after sequential second- and third- generation ALK-TKIs was reported to be G1202R-containing compound mutations [13,36]. On the other hand, not all compound ALK mutations are refractory to currently avail- able ALK-TKIs. For example, the sensitivity of lorlatinib against C1156Y/G1269A was observed in vitro [13], and the compound ALK C1156Y/L1198F mutation was reported to be resistant to ceritinib, alectinib, brigatinib, and lorlatinib but sensitive to crizotinib [36]. In addition, L1198-containing compound mutations are associated with the sensitivity to crizotinib [13] (Figure 1). It was recently reported that gilter- itinib, which is a TKI for treating relapsed or refractory FLT3- positive acute myeloid leukemia (AML), had shown

inhibitory effects on various single and compound muta- tions in vitro and in vivo, especially I1171N mutations (e.g. I1171N + F1174I and I1171N + L1198H) [94].

4.1.2. ALK-independent mechanisms (Off-target resistance) Activation of EGFR. Redaelli et al. reported that H3122 and H2228-derived lorlatinib-resistant cells showed activation of EGFR in vitro (H3122 and H2228 are NSCLC cell lines carrying different EML4/ALK variants [H3122, variant 1; H2228, variant 3a/b]) [95] Treatment of H3122-derived lorlati- nib-resistant cells with erlotinib resensitized the cells to lorla- tinib. In addition, upfront combined treatment with lorlatinib and erlotinib prevents the emergence of a resistant clone from H3122 cells. On the other hand, H2228-derived lorlatinib- resistant cells were resistant to the combined treatment with lorlatinib and EGFR-TKI; however, treatment with trametinib, which is a MEK inhibitor, in combination with lorlatinib and erlotinib showed remarkable growth inhibition [95]. TP53, NRAS-G12D, and MAP3K1 mutations. In the study that analyzed 20 lorlatinib-resistant cases, several co- occurring mutations were detected in addition to ALK resis- tance mutations; the most common mutation was TP53 muta- tion in 10 of the 20 cases [13]. In another retrospective study of 216 stage IIIB/IV patients with ALK-positive NSCLC, the PFS and OS of patients with TP53 mutations were inferior in com- parison to TP53 wild-type patients treated with chemotherapy and ALK-TKIs [96]. Since TP53 mutations has been reported to damage tumor suppressor functions as loss of function muta- tions [97], TP53 mutations might be associated with resistance to lorlatinib.
In the above study, which analyzed 20 lorlatinib-resistant cases, two potential drivers of ALK-independent resistance were identified: an activating NRAS-G12D mutation and a MAP3K1 mutation. Specifically, since the NRAS-G12D muta- tion was not detected in the patient’s pre-lorlatinib specimen, the NRAS-G12D mutation might mediate ALK-independent resistance against lorlatinib [13]. Neurofibromatosis type 2 (NF2) loss-of-function mutations. An NF2 loss of function mutation was identified as a novel mechanism of bypassing resistance to lorlatinib based on the analysis of a patient-derived cell line [11]. NF2 was reported to encode the merlin protein, which is an impor- tant tumor suppressor that is involved in the regulation of the PI3K-AKT-mTOR pathway through inhibition of mTOR [98]. These results suggested that mTOR inhibitors combined with lorlatinib could overcome the resistance to lorlatinib mediated by the NF2-loss of function. EMT. The analysis of the two patient-derived cell lines before and after lorlatinib treatment revealed that EMT mediated resistance to lorlatinib and was sensitive to dual SRC and ALK inhibition [11]. The lorlatinib-resistant cells lacked the expression of E-cadherin and had high N-cadherin, snail and vimentin expression levels; these findings were not observed in the pre-lorlatinib specimens. In addition, the SRC inhibitor

saracatinib, in combination with lorlatinib, showed a potent synergistic effect on lorlatinib-resistant cells [11]. Transformation to neuroendocrine carcinoma. In the case of a patient with ALK-positive NSCLC progressing on three-lines of ALK-TKIs, both transformation to a neuroendocrine carcinoma and acquisition of ALK L1196M was identified after development of acquired resistance to lorlatinib [99].

5. Immunotherapy strategies
The role of immunotherapy in the treatment of ALK-positive NSCLC has not been clearly established. To date, the efficacy of immune checkpoint inhibitor is limited against ALK-positive NSCLC. Subgroup analysis of pretreated patients with ALK fusion in the main immune oncology clinical trials revealed a strongly reduced benefit from monotherapy with immune checkpoint inhibitor [100–102]. Recently, the result of IMMUNOTARGET, which was a multicenter registry of patients harboring driver mutations and receiving immune oncology monotherapy, was reported [103]. Among the evaluated 21 ALK-positive patients, the objective response rate was 0%, and the median PFS was only 2.5 months (95% CI: 1.5–3.7 months) [103]. Retrospective studies reported the NSCLC with driver mutations such as EGFR and ALK being generally associated with a ‘cold’ immune tumor microenvironment, thus substan- tially impairing clinical responses to immune checkpoint inhi- bitor [104,105]. Although some trials of ALK-TKI and immune checkpoint inhibitor combination demonstrated certain anti- tumor activity in ALK-positive patients with acceptable safety profile, further analysis is needed to use immune checkpoint inhibitor for ALK-positive NSCLC.

6. Discussion
We reviewed the mechanisms of acquired ALK-TKI resistance, the methods for monitoring its appearance, and current and future efforts to develop treatment strategies to overcome resistance. Despite notable advances in tackling ALK-TKI resis- tance, resistance mechanisms have mainly been demonstrated in preclinical models. Although some resistance mechanisms are based on clinical data, those are all retrospective, and there are no prospective data to validate these different mod- els. Thus, at the present time, with the exception of the other ALK-TKIs, there are few approved regimens for patients with disease progression on second- or third-generation ALK-TKIs (Table 2).
To improve the prognosis of ALK-positive NSCLC, it is necessary to overcome and prevent those resistance mechan- isms. With regard to ALK-dependent mechanisms, lorlatinib has shown good efficacy in ALK-TKI-resistant cases, specifically among crizotinib-refractory cases (median PFS: not reached [95% CI: 11∙1–NR]) and cases with secondary mutations (11.0 months [95% CI, 6.9 to NR]) [23,24]. However, among second-generation ALK-TKI-refractory cases, the PFS was relatively short (median PFS: 5.5 months [95% CI: 2.7– 9.0]), even though secondary mutations were found in

Table 2. Clinical trials evaluating combinations of targeted therapies to address resistance mechanisms in patients with ALK-positive NSCLC.


7. Expert opinion
As described above, several mechanisms of second- and third-

identifier* Drug regimen Phase

population Status

generation ALK-TKI resistance have been reported. Some drugs that can overcome such resistance have been devel-

NCT02292550 Ceritinib + Ribociclib
(CDK4/6 i)
NCT02321501 Ceritinib + Everolimus
NCT03087448 Ceritinib + Trametinib

I/II without any
prior treatment

I without prior ALK-TKI
I/II with or without prior ALK- TKI


Ongoing Ongoing

oped, and several trials are ongoing to access their efficacy and safety. Clarifying the mechanisms of resistance is a key challenge to improving the prognosis of patients harboring resistance to next-generation ALK-TKIs. Despite notable advances in tackling therapy resistance in experimental set- tingsto date, there are few approved regimens for patients with disease progression on second- or third-generation ALK

NCT02521051 Alectinib + Bevacizumab I/II without any
prior treatment


inhibitors, especially for patients with compound mutations or
ALK-independent mechanisms of resistance.
Re-biopsy, which shows high sensitivity, is currently the

NCT03202940 Alectinib + Cobimetinib (MEKi)
NCT04005144 Brigatinib + Binimetinib (MEKi)

IB/II with prior alectinib
I with prior ALK- TKI

Ongoing Ongoing

method that is most commonly used to identify these mechan-
isms of resistance, and which has revealed many mechanisms of resistance; however, re-biopsy is invasive for the patient, and it is

NCT04227028 Brigatinib + Bevacizumab Ib with prior ALK-
NCT02584634 Lorlatinib + Avelumab II without any



often difficult to access certain tumor sites or to obtain adequate
amounts of tumor cells, especially among cases harboring resis- tance against ALK-TKIs. In addition, re-biopsy can only reveal the

NCT04292119 Lorlatinib + Crizotinib
(METi) or Binimetinib (MEKi)


I/II with prior ALK- TKI


mechanism of resistance in the part of the tumor that was biopsied. A liquid biopsy approach can be applied repeatedly in a noninvasive manner, and the analysis of the circulating

ALK, anaplastic lymphoma kinase; i, inhibitor; CDK, cyclin-dependent kinase; mTOR, mammalian target of rapamycin, TKI, tyrosine kinase inhibitor
*Further details on trials with NCT numbers can be accessed at the clinicaltrials. gov website.

approximately 50% of patients after second-generation ALK- TKI failure [23]. In addition, it was reported that the selection of ALK-TKI based on ALK-resistance mutations was associated with a high ORR and a relatively short PFS [106]. This is probably because compound mutations emerge more easily under the existence of ALK-resistance mutations, and com- pound mutations often mediate resistance to lorlatinib [13]. It may be possible to prevent the emergence of ALK-resistance mutations with upfront treatment using lorlatinib. Recently, the superiority of lorlatinib to crizotinib in first-line treatment was demonstrated with respect to PFS in a randomized phase III trial (the CROWN trial) [93]. The subsequent results of the CROWN trial will be helpful for deciding an optimal ALK-TKI sequential strategy.
Multiple ALK-independent resistance mechanisms with the heterogeneous evolution of tumors might be associated with the development of such resistance. In some ongoing trials that aim to overcome ALK-independent resistance, combina- tion treatment is expected to inhibit the activation bypass or parallel pathways (Table 1). With the development of NGS, it is expected that there will be more chances to clarify the mechanisms of resistance.
For the curative treatment of ALK-positive NSCLC, it is essential to understand the multi-factorial biological basis of resistance to ALK-TKI. The optimal measurement and use of biomarkers to identify or predict resistance and the develop- ment of new treatments that overcome such resistance are important for achieving this goal.

tumor DNA obtained by liquid biopsy can provide insight into
the extent of intra-tumoral heterogeneity [107,108]. Thus, liquid biopsy has the potential to noninvasively complement the find- ings of re-biopsy and may be useful for clarifying the hetero- geneous mechanisms of resistance.
In addition, the use of noninvasive circulating tumor DNA monitoring has also been considered for continuous monitor- ing to detect the emergence of resistance mechanisms. In EGFR-mutated NSCLC, an increased frequency of T790M muta- tions was detected based on circulating tumor DNA before the onset of clinical resistance to EGFR-TKIs [109,110]. This finding suggests that the selection of optimal sequential treatment using liquid biopsy before RECIST-defined disease progression (RECIST-PD) might prevent the emergence of several resis- tance mechanisms and overcome the challenge of the activa- tion of several off-target pathways.
A key weakness of liquid biopsy is its low sensitivity because some tumors do not shed circulating tumor DNA into the plasma. NGS is also associated with high cost and a long turnaround time. The development of highly sensitive methods, including allele-specific polymerase chain reaction (PCR), digital PCR (dPCR), and other modalities is expected in parallel with the generalization of NGS so that patients with ALK-TKI resistance can receive optimal targeted therapy.
The ALK-positive NSCLC population is not large, and it might be difficult to perform a randomized trial – based on the results of liquid biopsy – to evaluate the efficacy of tailored sequential treatment for cases with ALK-TKI resistance. It may be useful to conduct an umbrella study for TKI-resistant cases with rare driver mutations (e.g. RET, ROS1, and BRAF). From this author’s per- spective, because of the remarkable development of NGS, tai- lored sequential treatment based on the results of liquid biopsy can be expected to be standardized within a few years.

This paper received no funding.

Declaration of interest
The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Reviewer disclosures
Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

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