Monitoring therapeutic response and resistance with liquid biopsy

Introduction

Lung cancer is the leading cause of cancer-related death in industrialized countries and one of the most common cancers in the world. A total of 29,638 new cases estimated per year in Spain in the general population in 2020 and 22,153 deaths in 2019. The most common type of lung cancer is non-small cell lung cancer (NSCLC), which accounts for 80–90%. Approximately 3–7% of NSCLC patients have a genomic rearrangement of the anaplastic lymphoma kinase (ALK) gene (1).

ALK is a tyrosine kinase encoded on chromosome 2 and is primarily involved in developmental processes and expressed at low levels in adults. The first and the most prevalent fusion partner involved a fusion between the echinoderm microtubule-associated protein-like 4 (EML4) gene and the ALK tyrosine kinase domain (2). Other additional ALK fusion partners have been described in NSCLC that are believed to result in aberrant signaling and oncogenic transformation. ALK rearrangements are more common among patients with adenocarcinoma histology, patients who have never smoked, and patients who have wild-type EGFR and KRAS.

The standard treatment algorithm for unselected NSCLC patients has historically involved front-line treatment with chemotherapy, however recent clinical studies have demonstrated that patients with ALK-positive locally advanced or metastatic NSCLC respond well to treatment with an ALK tyrosine kinase inhibitor (ALK-TKI), it is an effective targeted medicine for these patients and can significantly prolong their survival.

ALK-TKI

The therapeutic arsenal includes first-generation ALK-TKI: crizotinib and more recently second-generation ALK-TKI: ceritinib, ensartinib, alectinib and brigatinib and third-generation ALK-TKI: lorlatinib.

Crizotinib is a first-generation ALK-TKI. It is a selective small molecule inhibitor of ALK, hepatocyte growth factor receptor (HGFR), c-MET and ROS-1. In preclinical studies, crizotinib demonstrated a concentration-dependent inhibitory activity of the growth of cell lines and animal models (xenografts) carrying the ALK, c-MET and ROS-1, inducing a powerful cellular apoptosis of the same, what was correlated, from a pharmacodynamic point of view, with the inhibition of ALK fusion protein phosphorylation in tumors in vivo. It has been used as a first-line therapy for patients with ALK-positive locally advanced or metastatic NSCLC, demonstrating objective response rate (ORR) around60–70%, progression-free survival (PFS) around 8–11 months and specific improvements in quality of life with respect to treatments according to the results of the main phase III studies, PROFILE 1007 (3) and PROFILE 1014 (4,5).

PROFILE 1007 is a phase III, open-label, randomized clinical trial that evaluates the efficacy and safety of crizotinib for the treatment of patients with ALK-positive metastatic NSCLC, who had received previous systemic treatment for advanced disease versus standard of care. The full analysis population included 347 patients with ALK-positive advanced NSCLC as identified by FISH prior to randomization. One hundred seventy-three patients were randomized to crizotinib and 174 patients were randomized to chemotherapy (either pemetrexed or docetaxel). Crizotinib significantly prolonged PFS, the primary objective of the study, compared to chemotherapy as assessed by IRR. The median PFS was 7.7 months for the group treated with crizotinib versus 3.0 months for the group treated with chemotherapy. The PFS benefit of crizotinib was consistent across subgroups of baseline patient characteristics such as age, gender, race, smoking class, time since diagnosis, ECOG performance status, presence of brain metastases and prior EGFR TKI therapy. There was a numerical, but not statistically significant, improvement in overall survival (OS) for crizotinib vs. chemotherapy. The crossover was allowed.

In the PROFILE 1014 clinical trial it was evaluated the efficacy and safety of crizotinib for the treatment of patients with ALK-positive metastatic NSCLC, who had not received previous systemic treatment for advanced disease. The full analysis population included 343 patients with ALK-positive advanced NSCLC as identified by fluorescence in situ hybridization (FISH) prior to randomization: 172 patients were randomized to crizotinib and 171 patients were randomized to chemotherapy, 6 cycles of platinum doublet (pemetrexed + carboplatin or cisplatin). PFS was significantly longer with crizotinib,10.9 months, than with chemotherapy, 7.0 months. ORR were 74% and 45%, respectively (P<0.001).

The problem is that about 30% of patients who used crizotinib as initial treatment have primary resistance to it and some patients developed secondary resistance within 1–2 years. Further, about 40% developed metastasis in central nervous system (CNS) (6).

Other ALK-TKI were gradually introduced and approved for clinical application.

Ceritinib is a potent oral, ATP-competitive inhibitor of the tyrosine kinase domain of ALK. In addition, it inhibits insulin-like growth factor receptor 1 (IGF-1R), the insulin receptor (InsR) and ROS1. Ceritinib is 20 times more potent than crizotinib. Phase I trial demonstrated that ceritinib induced responses in up to 60% of ALK-positive NSCLC patients. Ceritinib is equally active in patients previously treated with crizotinib as in those who have not received this treatment. Likewise, it shows response in brain metastases in patients who have progressed to crizotinib. On the other hand, ceritinib appears to be active regardless of the acquired resistance mechanism. The FDA in 2014 granted accelerated approval of ceritinib as a treatment for patients diagnosed with advanced ALK-positive advanced NSCLC in a state of progression after treatment with crizotinib or in cases of intolerance to crizotinib. The phase III study, ASCEND-4 (7) compared ceritinib 750 mg/day versus platinum-based chemotherapy (cisplatin 75 mg/m² or carboplatin AUC 5–6 plus pemetrexed 500 mg/m²) every 3 weeks for four cycles followed by maintenance pemetrexed in patients with ALK-positive advanced NSCLC not previously treated. The ORR for patients who received ceritinib was 72.5% compared to 26.7% for the group of patients who received chemotherapy, and the PFS was statistically higher also for the group of patients who received ceritinib 16.6 months versus 8.1 months for patients who received chemotherapy (HR 0.55, 95% CI: 0.42–0.73, P<0.00001). The intracranial ORR determined by RECIST v1.1 was 72.7% with ceritinib vs. 27.3% with chemotherapy, while the PFS in patients without basal brain metastases was 26.3 months with ceritinib versus8.3 months with chemotherapy.

Alectinib is a highly selective inhibitor of ALK and has shown systemic and CNS superiority in the first line compared to crizotinib in two phase III studies. The J-ALEX study (8), conducted exclusively in Japan, included 207 patients with advanced ALK-positive NSCLC, who were chemotherapy-naïve or had received one previous chemotherapy regimen. This study confirmed a significant benefit in terms of PFS for alectinib 300 mg/12 hours (HR: 0.34; P<0.0001) compared to crizotinib. Recently it has been reported final PFS data and the second pre-planned interim analysis of OS and safety (9). Median follow-up was 42.4 months for alectinib and 42.2 months for crizotinib. At the final PFS analysis, alectinib continued to demonstrate superiority in independent review facility (IRF)-assessed PFS versus crizotinib in ALK-inhibitor-naïve ALK-positive NSCLC (HR 0.37, 95% CI: 0.26–0.52; median PFS 34.1 vs. 10.2 months crizotinib), with a favorable safety profile. At the second interim OS analysis, superiority of alectinib to crizotinib could not be concluded (stratified HR 0.80, 99.8799% CI: 0.35–1.82, stratified log-rank P=0.3860; median OS not reached alectinib vs. 43.7 months crizotinib). OS follow-up continues. The global phase III study ALEX (10), conducted in 303 patients with advanced ALK-positive NSCLC not previously treated, compared alectinib 600 mg/12 hours vs. crizotinib 250 mg/12 hours. With a follow-up of 18.6 months for alectinib and17.6 months for crizotinib, the PFS evaluated by an independent radiological committee (IRC) was 25.7 vs. 10.4 months (HR 0.50, P<0.001). An update of the results of this study, with an additional follow-up, confirmed the benefit in PFS documented by the investigator (34.8 vs.10.9 months, HR 0.43, and ORR 82.9 vs. 75.5%) for alectinib compared to crizotinib (11). OS data is still immature (37% of events). Median OS was not reached with alectinib versus 57.4 months with crizotinib (stratified HR 0.67, 95% CI: 0.46–0.98). The 5-year OS rate was 62.5% (95% CI: 54.3–70.8%) with alectinib and 45.5% (95% CI: 33.6–57.4%) with crizotinib, with 34.9% and 8.6% of patients still on study treatment, respectively. The OS benefit of alectinib was seen in patients with CNS metastases at baseline and those without (12).

Brigatinib is a TKI that targets ALK, c-ros oncogene 1 (ROS1) and insulin like growth factor 1 receptor (IGF 1R). Brigatinib inhibits autophosphorylation of ALK and ALK mediated phosphorylation of the downstream signaling protein STAT3 in vitro and in vivo assays.

The efficacy and safety assessment of brigatinib is based on three studies:

The study AP26113-11-101 (13) was a phase I/II study of the safety, tolerability, pharmacokinetics (PK) and preliminary antitumor activity of brigatinib in advanced malignancies, including ALK-positive NSCLC. A total of 137 patients with advanced cancer were enrolled, including 79 patients (58%) with ALK-positive NSCLC, of which 71 had been previously treated with crizotinib. A total of 50 ALK-positive NSCLC patients had brain metastases at baseline. The 90 mg/180 mg cohort included 25 patients with ALK-positive NSCLC previously treated with crizotinib. In this group, 19/25 patients (76.0%; 95% CI: 54.9–90.6%) had a confirmed objective response. The median time to response was 1.9 months (range, 1.2–6.0 months). The median PFS was 16.3 months (95% CI: 9.2–not reached). The median OS was not reached in this group. Of those patients with ALK-positive NSCLC previously treated with crizotinib in the 90/180 mg group, there were 18 evaluable patients with brain metastases at baseline, of which 8 (44.4%; 95% CI: 21.5–69.2%) had a confirmed response by IRC. The median intracranial duration of response was 11.4 months (95% CI: 5.6–11.4 months) and the median intracranial PFS was not reached.

Most crizotinib-treated patients with ALK-positive NSCLC eventually experience disease progression.

The study AP26113-13-201 (ALTA) (14) was a pivotal phase II study in patients with advanced ALK-positive NSCLC whose disease had progressed to crizotinib. A total of 222 patients were enrolled and randomized 1:1 to receive brigatinib 90 mg once daily (112 patients) or brigatinib 180 mg once daily with a 7-day lead-in at 90 mg (110 patients). Patients were stratified by CNS metastases and best response to crizotinib. 154 (69%) had baseline brain metastases and 164 (74%) had received prior chemotherapy. The primary end point was investigator-assessed confirmed ORR per Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1. Secondary end points included independent review committee (IRC)-assessed PFS, intracranial PFS (iPFS) and OS. The median follow-up was 19.6 versus 24.3 months. Investigator-assessed confirmed ORR was 45.5% (97.5% CI: 34.8–56.5%) for the 90 mg group compared to 56.4% (97.5% CI: 45.2–67.0) for the 90/180mg group. Confirmed CR by investigator assessment was also increased for the 90/180 mg group: 4.5% vs. 1.8%. The ORR by IRC was similar. The median time to response was 1.8 to 1.9 months. The updated estimate median investigator-assessed duration of response was 12.0 months (95% CI: 9.2–17.7 months) for patients in the 90 mg group and 13.8 months (95% CI: 10.2–19.3 months) for patients in 90/180 mg group. IRC assessed duration of response was in line. Median IRC-assessed PFS was 9.2 months (95% CI: 7.4–12.8 months) for the 90 mg group and 16.7 months (95% CI: 11.6–21.4 months) for the 90/180 mg group. PFS results assessed by investigators were also similar. Median OS was 29.5 months in the 90 mg once daily group (95% CI: 18.2–not reached) versus34.1 months (95% CI: 27.7–not reached) in the 90/180 mg group. Brigatinib showed promising efficacy in the CNS, IRC-confirmed intracranial ORR in patients with measurable baseline brain lesions was 50% (13 of 26) versus 67% (12 of 18); median duration of intracranial response was 9.4 versus 16.6 months. IRC-assessed iPFS was 12.8 versus 18.4 months (15).

The third study that evaluated the role of brigatinib was the AP26113-13-301 (ALTA-1L) (16), an open-label, phase III trial, comparing brigatinib 180 mg once daily, after 7-day lead-in period at 90 mg once daily, or crizotinib at a dose of 250 mg twice daily. The primary end point was PFS as assessed by blinded independent central review. Secondary end points included the ORR and intracranial response. A total of 275 patients, not previously treated with ALK-TKI but who may be chemotherapy-naïve or who may have received one previous chemotherapy regimen, were randomized. Recently it has been published the second interim analysis (150 events) (17), the median follow-up was 24.9 months in the brigatinib arm and months in the crizotinib arm. Brigatinib showed consistent superiority in BIRC-assessed PFS versus crizotinib (HR 0.49, 95% CI: 0.35–0.68; log-rank P<0.0001; median, 24.0 vs. 11.0 months, respectively). Investigator-assessed PFS HR was 0.43 (95% CI: 0.31–0.61), median PFS was 29.4 vs. 9.2 months. Brigatinib significantly delayed both CNS progression (without prior systemic progression) and systemic progression (without prior intracranial progression) compared to crizotinib.

Currently, two other phase III clinical trials are evaluating the value of lorlatinib (CROWN, NCT03052608) and ensartinib (eXalt3, NCT02767804) compared to crizotinib in patients with advanced ALK-positive NSCLC. In the phase III CROWN study compared lorlatinib and crizotinib in first-line treatment in 296 ALK-naïve NSCLC patients. Crossover was not allowed. PFS by BICR (blinded independent review committee) was significantly prolonged with lorlatinib vs. crizotinib (HR 0.28; 95% CI: 0.19–0.41; P<0.001, not reached median PFS with lorlatinib vs.9.3 months with crizotinib). Lorlatinib also improved RR by BIRC compared with crizotinib (76% vs. 58%, respectively) and intracranial activity RR among patients with measurable brain metastases (82% vs. 23%) (18).

Positive findings from the eXalt3 trial of ensartinib have been reported at the 2020 Virtual Presidential Symposium of the World Congress on Lung Cancer. Ensartinib is an ALK-TKI, with potency 10 times greater than crizotinib in enzymatic assays and broad preclinical activity in ALK resistance mutations. In a phase II trial ensartinib had activity and was well tolerated in patients with crizotinib-refractory, ALK-positive NSCLC, including those with brain metastases (19). The primary endpoint of the phase III trial of BIRC-assessed PFS in the ITT population was 25.8 months for the patients assigned to receive ensartinib versus 12.7 months for the patients who received crizotinib. OS is still immature. Ensartinib showed a favourable safety profile, serious treatment-related adverse events (TRAEs) occurred in 8% of the patients treated with ensartinib and 6% of those who received crizotinib.

Although crizotinib, ceritinib, alectinib and brigatinib are recommend in the first line setting by the NCCN guidelines, alectinib is considered the preferred option.

ALK-TKI resistance

In approximately 50% of patients the CNS is the first site of progression (20), suggesting inadequate penetration of crizotinib into the brain (i.e., pharmacologic failure) as the primary cause of resistance in these patients. Intra-tyrosine kinase secondary ALK mutations represents the main mechanism of resistance to second-generation ALK TKIs, reported in more than 50% of patients. The same patient may have two or more resistance patterns.

Although, a large part of the resistance mechanism still remains unknown, ALK-TKI resistance can be divided into primary resistance and secondary resistance.

Primary resistance or intrinsic resistance refers to the ineffective treatment from the start. About 25% of patients treated with crizotinib have primary resistance. It is speculated that the mechanism of primary resistance is closely related to ALK variations, such as pre-existing ALK polymorphisms, deletions, and mutations (21). Tumor heterogeneity can also lead to primary resistance. Studies have found that 5–8% of cancer cells in ALK-positive NSCLC patient contain EGFR mutation, which causes the failure of ALK-TKI. Another cause may be false-positive genotype.

Secondary resistance or acquired resistance refers to the recurrence of tumor or tumor progression after having achieved complete response or partial response to ALK-TKI (22). Secondary resistance can be divided into dominant and non-dominant.

Dominant secondary resistance mainly refers to ALK kinase domain mutation (29%) and increase of ALK gene copy number (9%), which accounts for one-third of crizotinib resistance by increasing the activity of tyrosine kinases. Most of the mutations currently found in the target ALK gene are mainly point mutation. C1156Y and L1196M were the first discovered mutant types, followed by L1152R, G1202R, G1269A, F1174L, 1151Tins, S1206Y, I1171T, D1203N, V1180L, etc. (23-26). The most common mutation of ALK kinase domain in the crizotinib resistance is L1196M and G1269A (27). Patients with L1196M are sensitive to treatment with second-generation ALK-TKI.

ALK resistance mutations were different after crizotinib or after treatment with second-generation ALK-TKI. ALK resistance mutations were present in over one-half of patients progressing to second-generation ALK-TKI and also the spectrum of ALK resistance mutations was different following progression on second-generation ALK-TKI compared to crizotinib. Most notably, ALK G1202R, which was present in only 2% of crizotinib-resistant biopsies, emerged as the most common ALK resistance mutation after treatment with second-generation ALK-TKI. While ALK G1202R was a common shared resistance mutation in each second-generation ALK, it is noteworthy that the spectrum of other ALK resistance mutations appeared to different across agents. For example, ALK mutations such as F1174L/C, C1156Y and L1196M were observed in several post-ceritinib biopsy specimens, alectinib also included I1171T/N/S, V1180L and L1196M while E1210K, D1203N and S1206Y/C ALK mutations appears to be more frequent with brigatinib (24).

Non-dominant secondary resistance includes ALK signal bypass activation, and tumor heterogeneity. Signal bypass activation refers to the activation of other carcinogenic drivers and results in 20% of the cases of crizotinib resistance (28). The most common driven genes include EGFR and KRAS mutations, EGFR phosphorylation and c-KIT amplification. Other mechanisms is transformation to small cell lung cancer (SCLC), it involves tumor suppressor genes such as, Rb1 and p53, and this has been observed in a patient with crizotinib treatment failure (29) and in a patient with alectinib treatment failure (30).

Liquid biopsy

At present, the follow-up of patients with lung cancer during treatment mainly depends on the imaging tests and pathological biopsy. However, pathological biopsy is an invasive test and is not always possible to perform. Liquid biopsy emerges as a useful tool to provide genetic landscape of cancer lesions (31). The most common biomarkers of liquid biopsy are circulating tumor cells (CTCs), circulating free DNA (cfDNA), exosomal RNA or tumor platelets and they offer the potential for early diagnosis, identification of therapeutic targets, real-time monitoring of therapies and resistance mechanisms. Therefore, liquid biopsy might be the most convenient in the clinical setting particularly when tissue is unavailable or as a first screening for assessing resistance. It has different advantages such as simplicity, minimally invasive, real time and reproducibility.

The CTCs are the tumor cells that fall off from primary lesions or metastases of tumor ad then enter into the blood circulation (31). The detection rate of CTCs is low; only one CTC can be detected in an average of 10⁵–10⁷ monocytes in the peripheral blood of patients with advanced tumors (32). There are several methodologies for CTC detection, counting and characterization. The CellSearch system which is approved by FDA. In addition, Digital droplet polymerase chain reaction combined with next-generation sequencing (NGS) is currently used to detect and sequence genes in CTCs. CTCs presence and counting has been associated with worse prognosis in ALK-positive NSCLC patients (33). Moreover, some researchers have reported that ALK status can be effectively assessed in CTCs isolated from NSCLC patients (34,35). However, sample sizes of the aforementioned studies are rather small and results from larger cohort are needed in order to determine the clinical utility of this approach in NSCLC patients harboring an ALK translocation.

Circulating free DNA (cfDNA) is defined as the tissue-specific DNA fragment that is released into the blood (36). Circulating tumor DNA (ctDNA) refers to the cfDNA that is secreted by tumor cells or released after apoptosis and necrosis (37). The ctDNA levels in cancer patients are positively correlated with tumor progression, and the half-life is generally about 2 hours. While ctDNA has been widely used for the detection of EGFR activating mutations in NSCLC patients (38-40), less progress has been made regarding non-invasive ALK testing. Nonetheless, ALK rearrangements can be detected through ctDNA analysis (41,42).

Noteworthy, in a cohort of 88 consecutive patients with 96 plasma-detected ALK fusions McCoach et al. demonstrate that comprehensive cfDNA NGS (Guardant360) provides a noninvasive means of detecting ALK fusions and characterizing resistance mechanism on progression (43). Nevertheless, ALK rearrangements are difficult to detect since these alterations involve a large number of base pairs and cfDNA is typically fragmented. In addition, breakpoints at DNA levels are different in each case. Therefore a negative result should be taken with caution. Conversely, point mutations in ALK locus are easier to detect. In this way the detection of resistance mutations in ALK locus, upon ALK-TKI failure is of particular interest in order to optimize the sequencing of ALK-TKI maximizing patient’s survival. Several cohorts describing the resistance mechanisms ALK-TKI using liquid biopsies have been described (44,45). The clinical utility of longitudinal plasma genotyping in monitoring the evolution of ALK-positive NSCLC patients have been also evaluated (46). In this way, our group has recently published a case report where we illustrate the usefulness of plasma NGS profiling for detecting drug resistance mechanism upon disease progression in NSCLC patients with EML4-ALK rearrangement (47).

On the other hand, disease burden has been correlated not only with the appearance of resistance mutations but with the quantitative values of the allelic frequency (AF) of plasma alterations (48). Similarly, it has been recently reported that ctDNA quantification is of prognostic significance, in terms of PFS, in ALK-positive patients (49).

Exosomes are extracellular vesicles (EVs) with a diameter of 30-120nm, the surface of which is made up of lipid bilayer vesicles, containing DNA, mRNA, miRNA, protein and other genetic materials (50). Exosomes have important implications in resistance testing, and it may be more valuable than the CTCs and the ctDNA. The concentration of exosomes in peripheral blood is higher than that of CTCs, and it can be detected in urine, saliva, cerebrospinal fluid, semen, milk, pleural effusion, ascites and other body fluids in addition to serum or plasma (51). Exosomes arose as a promising tool for ALK fusion testing since fusion detection at the RNA level is feasible. Of note, RNA inside EVs is protected from degradation and therefore is susceptible of being subjected to analysis. Reclusa et al. recently reported the identification of EML4-ALK translocation exosomal RNA derived from NSCLC patients (52). In this study only 19 patients with plasma samples were included (16 of them harboring an ALK fusion) but it opens the door for the development of non-invasive ALK fusion testing strategies based on EVs isolation.

Finally, tumor-educated platelets (TEPs) are emerging as promising biomarker source. Tumors are known to educate platelets by altering its RNA profile. The analysis RNA obtained from TEPs has been demonstrated to detect early- and late-stage NSCLC (53). TEPs may offer certain advantages over other non-invasive analytes, including their abundance and easy isolation, high-quality RNA obtained from them. It has been reported that non-invasive ALK testing can be performed using TEPs (53,54). However, larger studies are needed to further support the use of TEPs in the management of ALK-positive NSCLC patients.

Other body fluids like urine, saliva and pleural effusion have been used on the diagnosis of NSCLC. There are many studies on EGFR-TKI resistance surveillance, however, more studies and experiments are expected to confirm their viability in ALK-TKI resistance surveillance.

Discussion and conclusions

Liquid biopsy is applicable to the surveillance of all lung cancer patients, can predict tumor progression and plays an important role in warning resistance. This can help us prolong the survival and avoid side effects caused by ineffective treatment. In advanced ALK-positive NSCLC, alectinib has shown the best balance of efficacy and safety and is the current preferred first-line treatment option. Lorlatinib is approved in advanced ALK-positive NSCLC patients whose disease has progressed to prior second-generation ALK TKIs. It is very interesting to known the molecular profiling at the time of disease progression to second and third-generation ALK TKIs because this may provide relevant information to define genomic-driven therapeutic sequences. Even though, the use of ALK-TKI according to the resistance mechanism, at the time of progression, seems the most appropriate, blinded treatment decisions are the most common in the clinic.

Acknowledgments

We appreciate the kind invitation to contribute to the PCM Series on “ALK and ROS-1 NSCLC patients treatment approach based on genomic profile by liquid biopsy” edited by Drs. Jesús Corral, Laura Mezquita and Ernest Nadal.

Funding: None.

Footnote

Provenance and Peer Review: This article was commissioned by the Guest Editors (Jesús Corral, Laura Mezquita and Ernest Nadal) for the series “ALK and ROS-1 NSCLC Patients Treatment Approach Based on Genomic Profile by Liquid Biopsy” published in Precision Cancer Medicine. The article has undergone external peer review.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/pcm-20-46). The series “ALK and ROS-1 NSCLC Patients Treatment Approach Based on Genomic Profile by Liquid Biopsy” was commissioned by the editorial office without any funding or sponsorship. Dr. AR serves as an unpaid editorial board member of Precision Cancer Medicine from Aug 2020 to Jul 2022. Dr. VCJ reports honoraria as speaker and consultant on advisory boards from: ROCHE, BMS, MSD, ASTRAZENECA, BOEHRINGER and TAKEDA. Dr. MP reports honoraria as speaker and consultant on advisory boards from: ROCHE, BMS, MSD, TAKEDA and LILLY.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

References

1. Sasaki T, Rodig SJ, Chirieac LR, et al. The biology and treatment of EML4-ALK non-small cell lung cancer. Eur J Cancer 2010;46:1773-80. [Crossref] [PubMed]
2. Soda M, Choi YL, Enomoto M, et al. Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer. Nature 2007;448:561-6. [Crossref] [PubMed]
3. Shaw AT, Kim D-W, Nakagawa K, et al. Crizotinib versus chemotherapy in advanced ALK-positive lung cancer. N Engl J Med 2013;368:2385-94. [Crossref] [PubMed]
4. Solomon BJ, Mok T, Kim DW, et al. First-line crizotinib versus chemotherapy in ALK-positive lung cancer. N Engl J Med 2014;371:2167-77. [Crossref] [PubMed]
5. Solomon BJ, Kim DW, Wu YL, et al. Final Overall Survival Analysis From a Study Comparing First-Line Crizotinib Versus Chemotherapy in ALK-Mutation-Positive Non-Small-Cell Lung Cancer. J Clin Oncol 2018;36:2251-8. [Crossref] [PubMed]
6. Ou SHI, Jänne PA, Bartlett CH, et al. Clinical benefit of continuing ALK inhibition with crizotinib beyond initial disease progression in patients with advanced ALK-positive NSCLC. Ann Oncol 2014;25:415-22. [Crossref] [PubMed]
7. Soria JC, Tan DSW, Chiari R, et al. First-line ceritinib versus platinum-based chemotherapy in advanced ALK-rearranged non-small-cell lung cancer (ASCEND-4): a randomised, open-label, phase 3 study. Lancet 2017;389:917-29. [Crossref] [PubMed]
8. Hida T, Nokihara H, Kondo M, et al. Alectinib versus crizotinib in patients with ALK-positive non-small-cell lung cancer (J-ALEX): an open-label, randomised phase 3 trial. Lancet 2017;390:29-39. [Crossref] [PubMed]
9. Nakagawa K, Hida T, Nokihara H, et al. Final progression-free survival results from the J-ALEX study of alectinib versus crizotinib in ALK-positive non-small-cell lung cancer. Lung Cancer 2020;139:195-9. [Crossref] [PubMed]
10. Peters S, Camidge DR, Shaw AT, et al. Alectinib versus Crizotinib in Untreated ALK-Positive Non-Small-Cell Lung Cancer. N Engl J Med 2017;377:829-38. [Crossref] [PubMed]
11. Camidge DR, Dziadziuszko R, Peters S, et al. Updated Efficacy and Safety Data and Impact of the EML4-ALK Fusion Variant on the Efficacy of Alectinib in Untreated ALK-Positive Advanced Non-Small Cell Lung Cancer in the Global Phase III ALEX Study. J Thorac Oncol 2019;14:1233-43. [Crossref] [PubMed]
12. Mok T, Camidge DR, Gadgeel SM, et al. Updated overall survival and final progression-free survival data for patients with treatment-naive advanced ALK-positive non-small-cell lung cancer in the ALEX study. Ann Oncol 2020;31:1056-64. [Crossref] [PubMed]
13. Gettinger SN, Bazhenova LA, Langer CJ, et al. Activity and safety of brigatinib in ALK-rearranged non-small-cell lung cancer and other malignancies: a single-arm, open-label, phase 1/2 trial. Lancet Oncol 2016;17:1683-96. [Crossref] [PubMed]
14. Kim DW, Tiseo M, Ahn MJ, et al. Brigatinib in Patients With Crizotinib-Refractory Anaplastic Lymphoma Kinase-Positive Non-Small-Cell Lung Cancer: A Randomized, Multicenter Phase II Trial. J Clin Oncol 2017;35:2490-8. [Crossref] [PubMed]
15. Huber RM, Hansen KH, Paz-Ares Rodríguez L, et al. Brigatinib in Crizotinib-Refractory ALK+ NSCLC: 2-Year Follow-up on Systemic and Intracranial Outcomes in the Phase 2 ALTA Trial. J Thorac Oncol 2020;15:404. [Crossref] [PubMed]
16. Camidge DR, Kim HR, Ahn MJ, et al. Brigatinib versus Crizotinib in ALK-Positive Non-Small-Cell Lung Cancer. N Engl J Med 2018;379:2027-39. [Crossref] [PubMed]
17. Camidge DR, Kim HR, Ahn MJ, et al. Brigatinib Versus Crizotinib in Advanced ALK Inhibitor-Naive ALK-Positive Non-Small Cell Lung Cancer: Second Interim Analysis of the Phase III ALTA-1L Trial. J Clin Oncol 2020;38:3592-603. [Crossref] [PubMed]
18. Shaw AT, Bauer TM, de Marinis F, et al. First-Line Lorlatinib or Crizotinib in Advanced ALK-Positive Lung Cancer. N Engl J Med 2020;383:2018-29. [Crossref] [PubMed]
19. Yang Y, Zhou J, Zhou J, et al. Efficacy, safety, and biomarker analysis of ensartinib in crizotinib-resistant, ALK-positive non-small-cell lung cancer: a multicentre, phase 2 trial. Lancet Respir Med 2020;8:45-53. [Crossref] [PubMed]
20. Costa DB, Shaw AT, Ou SHI, et al. Clinical Experience With Crizotinib in Patients With Advanced ALK-Rearranged Non-Small-Cell Lung Cancer and Brain Metastases. J Clin Oncol 2015;33:1881-8. [Crossref] [PubMed]
21. Heuckmann JM, Balke-Want H, Malchers F, et al. Differential protein stability and ALK inhibitor sensitivity of EML4-ALK fusion variants. Clin Cancer Res 2012;18:4682-90. [Crossref] [PubMed]
22. Jackman D, Pao W, Riely GJ, et al. Clinical definition of acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors in non-small-cell lung cancer. J Clin Oncol 2010;28:357-60. [Crossref] [PubMed]
23. Toyokawa G, Seto T. Updated Evidence on the Mechanisms of Resistance to ALK Inhibitors and Strategies to Overcome Such Resistance: Clinical and Preclinical Data. Oncol Res Treat 2015;38:291-8. [Crossref] [PubMed]
24. Gainor JF, Dardaei L, Yoda S, et al. Molecular Mechanisms of Resistance to First- and Second-Generation ALK Inhibitors in. Cancer Discov 2016;6:1118-33. [Crossref] [PubMed]
25. Heuckmann JM, Hölzel M, Sos ML, et al. ALK mutations conferring differential resistance to structurally diverse ALK inhibitors. Clin Cancer Res 2011;17:7394-401. [Crossref] [PubMed]
26. Choi YL, Soda M, Yamashita Y, et al. EML4-ALK mutations in lung cancer that confer resistance to ALK inhibitors. N Engl J Med 2010;363:1734-9. [Crossref] [PubMed]
27. Doebele RC, Pilling AB, Aisner DL, et al. Mechanisms of resistance to crizotinib in patients with ALK gene rearranged non-small cell lung cancer. Clin Cancer Res 2012;18:1472-82. [Crossref] [PubMed]
28. Boland JM, Jang JS, Li J, et al. MET and EGFR mutations identified in ALK-rearranged pulmonary adenocarcinoma: molecular analysis of 25 ALK-positive cases. J Thorac Oncol 2013;8:574-81. [Crossref] [PubMed]
29. Cha YJ, Cho BC, Kim HR, et al. A Case of ALK-Rearranged Adenocarcinoma with Small Cell Carcinoma-Like Transformation and Resistance to Crizotinib. J Thorac Oncol 2016;11:e55-8. [Crossref] [PubMed]
30. Takegawa N, Hayashi H, Iizuka N, et al. Transformation of ALK rearrangement-positive adenocarcinoma to small-cell lung cancer in association with acquired resistance to alectinib. Ann Oncol 2016;27:953-5. [Crossref] [PubMed]
31. Chen Y, Guo W, Fan J, et al. The applications of liquid biopsy in resistance surveillance of anaplastic lymphoma kinase inhibitor. Cancer Manag Res 2017;9:801-11. [Crossref] [PubMed]
32. Gupta GP, Massagué J. Cancer metastasis: building a framework. Cell 2006;127:679-95. [Crossref] [PubMed]
33. Allan AL, Keeney M. Circulating tumor cell analysis: technical and statistical considerations for application to the clinic. J Oncol 2010;2010:426218. [Crossref] [PubMed]
34. Provencio M, Pérez-Callejo D, Torrente M, et al. Concordance between circulating tumor cells and clinical status during follow-up in anaplastic lymphoma kinase (ALK) non-small-cell lung cancer patients. Oncotarget 2017;8:59408-16. [Crossref] [PubMed]
35. Ilie M, Long E, Butori C, et al. ALK-gene rearrangement: a comparative analysis on circulating tumour cells and tumour tissue from patients with lung adenocarcinoma. Ann Oncol 2012;23:2907-13. [Crossref] [PubMed]
36. Kulasinghe A, Lim Y, Kapeleris J, et al. The Use of Three-Dimensional DNA Fluorescent In Situ Hybridization (3D DNA FISH) for the Detection of Anaplastic Lymphoma Kinase (ALK) in Non-Small Cell Lung Cancer (NSCLC) Circulating Tumor Cells. Cells 2020;9:1465. [Crossref] [PubMed]
37. Shaw JA, Stebbing J. Circulating free DNA in the management of breast cancer. Ann Transl Med 2014;2:3. [PubMed]
38. Jahr S, Hentze H, Englisch S, et al. DNA fragments in the blood plasma of cancer patients: quantitations and evidence for their origin from apoptotic and necrotic cells. Cancer Res 2001;61:1659-65. [PubMed]
39. Provencio M, Torrente M, Calvo V, et al. Dynamic circulating tumor DNA quantificaton for the individualization of non-small-cell lung cancer patients treatment. Oncotarget 2017;8:60291-8. [Crossref] [PubMed]
40. Provencio M, Torrente M, Calvo V, et al. Prognostic value of quantitative ctDNA levels in non small cell lung cancer patients. Oncotarget 2017;9:488-94. [Crossref] [PubMed]
41. Romero A, Serna-Blasco R, Alfaro C, et al. ctDNA analysis reveals different molecular patterns upon disease progression in patients treated with osimertinib. Transl Lung Cancer Res 2020;9:532-40. [Crossref] [PubMed]
42. Aguado C, Giménez-Capitán A, Karachaliou N, et al. Fusion gene and splice variant analyses in liquid biopsies of lung cancer patients. Transl Lung Cancer Res 2016;5:525-31. [Crossref] [PubMed]
43. McCoach CE, Blakely CM, Banks KC, et al. Clinical Utility of Cell-Free DNA for the Detection of ALK Fusions and Genomic Mechanisms of ALK Inhibitor Resistance in Non-Small Cell Lung Cancer. Clin Cancer Res 2018;24:2758-70. [Crossref] [PubMed]
44. Noé J, Lovejoy A, Ou SHI, et al. ALK Mutation Status Before and After Alectinib Treatment in Locally Advanced or Metastatic ALK-Positive NSCLC: Pooled Analysis of Two Prospective Trials. J Thorac Oncol 2020;15:601-8. [Crossref] [PubMed]
45. Dagogo-Jack I, Rooney M, Lin JJ, et al. Treatment with Next-Generation ALK Inhibitors Fuels Plasma ALK Mutation Diversity. Clin Cancer Res 2019;25:6662-70. [Crossref] [PubMed]
46. Dagogo-Jack I, Brannon AR, Ferris LA, et al. Tracking the Evolution of Resistance to ALK Tyrosine Kinase Inhibitors through Longitudinal Analysis of Circulating Tumor DNA. JCO Precis Oncol 2018;2018:PO.17.00160.
47. Sánchez-Herrero E, Blanco Clemente M, Calvo V, et al. Next-generation sequencing to dynamically detect mechanisms of resistance to ALK inhibitors in ALK-positive NSCLC patients: a case report. Transl Lung Cancer Res 2020;9:366-72. [Crossref] [PubMed]
48. Zhang EW, Dagogo-Jack I, Kuo A, et al. Association between circulating tumor DNA burden and disease burden in patients with ALK-positive lung cancer. Cancer 2020;126:4473-84. [Crossref] [PubMed]
49. Madsen AT, Winther-Larsen A, McCulloch T, et al. Genomic Profiling of Circulating Tumor DNA Predicts Outcome and Demonstrates Tumor Evolution in ALK-Positive Non-Small Cell Lung Cancer Patients. Cancers 2020;12:947. [Crossref] [PubMed]
50. Taverna S, Giallombardo M, Gil-Bazo I, et al. Exosomes isolation and characterization in serum is feasible in non-small cell lung cancer patients: critical analysis of evidence and potential role in clinical practice. Oncotarget 2016;7:28748-60. [Crossref] [PubMed]
51. Vlassov AV, Magdaleno S, Setterquist R, et al. Exosomes: current knowledge of their composition, biological functions, and diagnostic and therapeutic potentials. Biochim Biophys Acta 2012;1820:940-8. [Crossref] [PubMed]
52. Reclusa P, Laes JF, Malapelle U, et al. EML4-ALK translocation identification in RNA exosomal cargo (ExoALK) in NSCLC patients: a novel role for liquid biopsy. Transl Lung Cancer Res 2019;8:S76-8. [Crossref]
53. Best MG, Sol N, In ’t Veld SGJG, et al. Swarm Intelligence-Enhanced Detection of Non-Small-Cell Lung Cancer Using Tumor-Educated Platelets. Cancer Cell 2017;32:238-252.e9.
54. Nilsson RJA, Karachaliou N, Berenguer J, et al. Rearranged EML4-ALK fusion transcripts sequester in circulating blood platelets and enable blood-based crizotinib response monitoring in non-small-cell lung cancer. Oncotarget 2016;7:1066-75. [Crossref] [PubMed]