Approximately 3-5% of tumors in patients with advanced non-small cell lung cancer (aNSCLC) harbor rearrangements of the anaplastic kinase lymphoma gene (ALK) (1, 2). This is a unique aNSCLC subpopulation that mostly consists of young individuals, with no or limited history of smoking, and an adenocarcinoma histology. Although ALK fusion is a rare phenomenon, it shouldn’t be neglected considering the high prevalence of lung cancer overall, and the availability of several effective targeted treatment options (3–5).

The presence of ALK rearrangement results in tumor susceptibility to ALK tyrosine kinase inhibitors (ALK TKIs) (6). Crizotinib, a TKI of ALK, tyrosine-protein kinase Met (c-met), and ROS proto-oncogene 1 (ROS1) kinases (7), was the first ALK inhibitor to replace the standard chemotherapy in the 1^st-line treatment of aNSCLC harboring an ALK fusion, providing the significant advantage of this therapy in terms of the progression-free survival - according to the results of the PROFILE 1014 trial (8). Since then, newer 2^nd- and 3^rd-generation ALK TKIs (e.g., alectinib, ceritinib, brigatinib, ensartinib and lorlatinib) were implemented into the management of ALK- rearranged aNSCLC - first in the post-progression setting (9–11), and later on - in the 1^st- line setting - that based on the results of the ALEX trial (12), the ALTA-1L trial (13), the ASCEND-4 trial (14), and the CROWN trial (15).

The questions of ALK TKIs sequencing and optimal treatment strategy following the disease progression on specific ALK TKIs remain open, since these have never been evaluated in a randomized controlled clinical trial (16). Treatment decisions, however, can be guided by the acquired resistance mechanisms responsible for the disease progression during systemic treatment. The mechanisms of acquired resistance to ALK TKIs primarily include development of secondary resistant mutations in the ALK kinase domain occurring in 25-66% of patients (17–21). Of those, G1202R/del mutations predominate (42-53% of cases), while other ALK mutation types responsible for the development of secondary resistance to ALK TKIs are: L1196M, F1174X, G1269A, L1196M, and I1171X (18, 21). Moreover, sequential treatment with increasingly potent ALK TKIs may promote acquisition of treatment-refractory compound ALK mutations (21, 22). Off-target mechanisms of resistance to ALK TKIs involve up-regulation of bypass signaling pathways, such as epidermal growth factor receptor (EGFR), c-met, KIT proto-oncogene (KIT), insulin-like growth factor 1 receptor (IGF-1R), proto-oncogene SRC (SRC), MEK/ERK and others (17, 18, 23, 24). SCLC transformation has been described as a resistance mechanism to ALK TKIs as well (25, 26).

Since ALK resistance mutations appear to be the predominant mechanism of resistance to ALK TKIs, there is a clear rationale for its targeting. Moreover, it has been demonstrated that the presence of an ALK resistant mutation following the progression on 1^st- and 2^nd-generation ALK TKIs in ALK-rearranged aNSCLC is associated with better lorlatinib efficacy (19). However, different 2^nd- and 3^rd-generation ALK TKIs appear to have different in vitro activity against specific ALK resistant mutations, which, therefore, represents the rationale for identifying the underlying ALK resistant mutation subtype before making the decision regarding the next line of systemic treatment.

In our study, we prospectively assessed the impact of comprehensive genomic profiling (CGP) on the decision-making process in patients with ALK-rearranged aNSCLC following progression on 2^nd- and 3^rd-generation ALK TKIs.

This is the first prospective study illustrating the value of CGP and molecular assessment of acquired resistance mechanisms in treatment decision-making process in ALK-rearranged aNSCLC patients.

According to our observation, CGP performed at the time of progression on 2^nd- and 3^rd-generation ALK TKIs, has altered treatment recommendation in one third of cases - which confirmed the initial hypothesis and, overall, appeared to be a clinically meaningful result. In cases the initiation of the NLST was postponed until getting NGS results, the proportion of patients in whom the treatment recommendation has changed was even higher (50%) - pointing to potentially larger effect of genomic assessment on the decision-making process.

Importantly, the median CGP TAT was only 2.9 weeks [IQR, 2.4-4.4] which seems acceptable considering the CGP impact on treatment decision. In our cohort, only one patient has died while waiting for the CGP results. This fact emphasizes the need to assess further the phenomenon of clinical deterioration attributable to rapid disease progression, and the potential adverse effect of the CGP in this association.

Looking into the specific treatment recommendation changes following the receival of the CGP results, we observed increase in the proportion of recommendations on lorlatinib, platinum-based chemotherapy, and crizotinib. There were three clinical scenarios which seemed to have a biologic rationale behind the treatment decision alteration. The 1^st clinical scenario was switching to a different targeted treatment following the diagnosis of another potentially targetable molecular aberration, or a bypass pathway activation, such as switching from ALK TKI to c-met TKI following the diagnosis of c-met amplification. Indeed, c-met alterations represent one of the most common mechanisms responsible for acquired resistance to osimertinib in EGFR mutant aNSCLC (10-25%) (29), and to ALK TKIs in ALK-rearranged aNSCLC (15%) (24). Moreover, c-met inhibition has been associated with objective response rate of 30% and median duration of response of 7.9 months in c-met-amplified aNSCLC patients following progression on 3^rd-generation EGFR TKIs (30). Additionally, there are several case reports suggesting that met-inhibition may overcome c-met-driven resistance in ALK-positive aNSCLC (24, 31, 32). The 2^nd clinical scenario of treatment decision alteration in our cohort was switching from brigatinib to lorlatinib following the diagnosis of ALK G1202R mutation – which is justified by the high activity of lorlatinib in tumors harboring this ALK resistant mutation (19). The 3^rd clinical scenario in our study included switching from ALK TKIs to platinum-based chemotherapy in the absence of ALK resistant mutation - which, again, seems reasonable considering modest next-generation ALK TKI activity in patients without ALK resistant mutations following progression on prior ALK TKIs (19). Specifically, a positive correlation between presence of ALK resistant mutations, their type, and outcomes with lorlatinib have been reported in ALK-rearranged aNSCLC patients following failure of a 2^nd-generation ALK TKI. It remains unknown, however, whether similar correlation is true for brigatinib. Moreover, no comparative clinical studies have been done or planned to be done in order to explore the comparative efficacy of the two agents in correlation with the molecular biomarkers in this clinical setting.

In three additional clinical scenarios which prompted treatment decision changes in our study, it was hard to explain the physician’s decision from the biologic perspective. For instance, presence of CDKN2A/B loss or mutation was anticipated to alter the decision towards cyclin-dependent kinase 4/6 (CDK4/6) inhibitors, however, it was not the case. Having said that, it should be emphasized that CDKN2A/B alterations are only rarely seen following progression on 2^nd/3^rd-generation ALK TKIs (17, 23, 33), and the majority of CDK4/6 inhibitors did not demonstrate a significant antitumor activity in aNSCLC (34–39). Although CDK4/6 inhibitors have demonstrated a myelo-preserving effect in conjunction with chemotherapy in advanced small-cell lung cancer, it did not appear to improve tumor control (40).

The prevalence of ALK resistant mutations (30%) and their distribution in our study were in line with the previously reported data in ALK-positive aNSCLC following treatment with 2^nd/3^rd-generation ALK TKIs (19, 23). Only one case of high-level c-met amplification was present in our cohort, while higher prevalence of c-met alterations (12-22%) has been reported in the literature (24). This discrepancy might be attributable to the lower proportion of patients progressing on lorlatinib in our cohort, and less so - to the technical limitations of the liquid biopsy assay. For instance, Dagogo-Jack et al. reported on higher prevalence of c-met amplification following treatment with 3^rd-generation ALK TKI, on one hand, and on the other hand - on high overall accuracy of liquid NGS as compared to tissue genotyping (24). The detection of CDKN2A/B alterations was not unique to our cohort either (33). Another interesting observation in our study was tissue versus liquid biopsy referral patterns. For instance, liquid biopsy was the preferred method of assessment - probably due to its simplicity and high patient advocacy, which reflected real-world physician and patient preferences. This pattern was also in line with the recently updated International Association for the Study of Lung Cancer (IASLC) guideline on liquid biopsy to discover molecular resistance mechanisms (41).

CGP performed at the time of progression on 2^nd/3^rd-generation ALK TKIs demonstrated a positive impact on NLST duration but did not affect the OS. Several factors might have attributed to that. First, some of the most expedient alterations in treatment recommendations were not implemented. Second, OS was the subject for the lead-time bias: i.e., those patients in whom the NLST was initiated following the receival of the CGP results, initiated the treatment later as opposed to patients in whom the NLST was started before the CGP results became available. Finally, higher proportion of patients in whom the treatment was initiated before receival of the CGP results appeared to harbor an ALK resistant mutation, which might have an impact on outcomes as well. The lack of the ability to demonstrate an impact of CGP on oncological outcomes remains the most significant limitation of our study, along with the small sample size. Another important study limitation is its non-randomized design allowing patient selection for immediate versus postponed treatment initiation based on the tempo of the disease.

Overall, the study has demonstrated the feasibility and the significant impact of the CGP on the decision-making process in ALK-rearranged aNSCLC following failure of 2^nd/3^rd-generation ALK TKIs. It remains to be seen whether such strategy affects oncological outcomes.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ Supplementary Material .

The studies involving human participants were reviewed and approved by the Helsinky ethics committee of the Rabin Medical Center (RMC), approval number: 0561-19-RMC. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.

Credit statement conceptualization: ED, AR, and AO. Methodology: ED, AR, and AO. Validation: ED. Formal analysis: TS. Investigation: ED. Resources: ED, AR, AO, LH, JD, DU, WK, AC, MM, AZ, JB, NR, SG, CO, AA, NP, and TS. Data Curation: ED. Writing - original draft: ED, AR, AO, and LH. Writing - review and editing: ED, AR, AO, LH, JD, DU, WK, AC, MM, AZ, JB, NR, SG, CO, AA, NP, and TS. Visualization: ED and LH. Supervision: ED. Project administration: ED. All authors contributed to the article and approved the submitted version.

Author TS was employed by the company Statistical Consulting Unit. Disclosure (all outside of the submitted work): AR reported personal fees from Roche, Astra Zeneca, Merck Sharpe & Dohme, Novartis, Takeda, Elli Lilly, support for attending meetings from Bristol Myers Squibb, Roche, Boehringer Ingelheim. AO reported advisory fees from Merck Sharpe & Dohme, Bristol Myers Squibb, Roche, Astra Zeneca, Novartis, Boehringer Ingelheim. Damien Urban reported personal and consulting fees from Roche, Merck Sharpe & Dohme, Takeda, Astra Zeneca, Rhenium Oncotest, Bristol Myers Squibb. MM reported consulting fees from Boehringer Ingelheim, Roche, Astra Zeneca, MSD, BMS, Abbvie, Takeda, Pomicell. AZ reported grants from Bristol Myers Squibb, personal fees from Roche, Merck Sharpe & Dohme, Bristol Myers Squibb, Astra Zeneca, Takeda. JB reported grants and personal fees from Merck Sharpe & Dohme, Bristol Myers Squibb, Astra Zeneca, Roche, Abbvie, Takeda, OncoHost, ImmuneAI, Bayer, Novartis. AA reported research funding from Bristol Myers Squibb, personal and consulting fees from Bristol Myers Squibb, Roche, Pfizer, Astra Zeneca, Takeda, Novartis. NP reported research funding from Bristol-Myers Squibb, Eli Lilly, Foundation Medicine, Gaurdant360, Merk, MSD, Novartis, NovellusDx, Pfizer, Roche, Takeda, IP: Volatile Organic Compounds For Detecting Cell Dysplasia And Genetic Alterations Associated With Lung Cancer, WO2012023138; Breath Analysis of Pulmonary Nodules, US20130150261 A1; Apparatus for treating a target site of a body, WO/2015/059646 - all outside of the submitted work. ED reported grants from Astra Zeneca, Boehringer Ingelheim, personal fees from Boehringer Ingelheim, Roche, Astra Zeneca, Pfizer, Merck Sharpe & Dohme, Bristol Myers Squibb, Novartis, Takeda, Sanofi, Merck Serono, Medison Pharma, Janssen Israel- all outside of the submitted work.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.