Deep RNA Sequencing Revealed Fusion Junctional Heterogeneity May Predict Crizotinib Treatment Efficacy in ALK-Rearranged NSCLC

IntroductionGene fusion variants in ALK-rearranged NSCLC may predict patient outcomes, but previous results have been inconclusive. Fusion isoforms coexisting in the same tumor may affect the efficacy of targeted therapy, but they have not been investigated.MethodsPatients with ALK-rearranged NSCLC who received crizotinib treatments were recruited. Precrizotinib tumor tissues were analyzed by the anchored multiplex polymerase chain reaction for targeted RNA sequencing. Kaplan-Meier and Cox regression were used to compare overall and progression-free survivals.ResultsOf the 51 studied subjects, EML4-ALK variant types v1, v2, v3, and others were detected in 23 (45.1%), five (9.8%), 19 (37.3%), and four patients (7.8%), respectively. Multiple EML4-ALK RNA isoforms were detected in 24 tumors (47.1%), and single isoform in 27 (52.9%). Most of the v3 tumors (16 of 19) harbored both v3a and v3b RNA isoforms. Multiple isoforms were also detected in eight non-v3 tumors (33.3% of all 24 multiple isoforms; five v1, two v5′, and one v2). Compared with patients with single isoform, those with multiple isoforms had worse progression-free (hazard ratio and 95% confidence interval: 2.45 [1.06–5.69]) and overall (hazard ratio [95% confidence interval]: 3.74 [1.26–11.13]) survivals after adjusting for potential confounders including variant type. Using the patient-derived H2228 cells known to express v3a and v3b, our single-cell polymerase chain reaction detected either v3a or v3b in most single cells. Treatment of H2228 cells by three ALK inhibitors revealed increased ratios of v3a-to-v3b expression over time.ConclusionsIntratumoral EML4-ALK isoforms may predict the efficacy of targeted therapy in ALK-rearranged NSCLC. Temporal changes of intratumoral fusion isoforms may result from differential selection pressures that a drug might have on one isoform over another. Larger studies on fusion heterogeneity using RNA sequencing are warranted.     

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

IntroductionAt the prior data cutoff (February 9, 2017) the ALEX trial showed superior investigator-assessed progression-free survival (PFS) for alectinib versus crizotinib in untreated, anaplastic lymphoma kinase (ALK)-positive, advanced NSCLC (hazard ratio = 0.47, 95% confidence interval: 0.34–0.65, p < 0.001). The median PFS in the alectinib arm was not reached versus 11.1 months with crizotinib. Retrospective analyses suggest that the echinoderm microtubule-associated protein-like 4 gene-ALK variant (EML4-ALK) may influence ALK-inhibitor treatment benefit. We present updated analyses, including exploratory subgroup analysis by EML4-ALK variant, after an additional 10 months’ follow-up (cutoff December 1, 2017).MethodsPatients were randomized to receive twice-daily alectinib, 600 mg, or crizotinib, 250 mg, until disease progression, toxicity, death, or withdrawal. PFS was determined by the investigators. Baseline plasma and tissue biomarker samples were analyzed by using hybrid-capture, next-generation sequencing to determine EML4-ALK variant.ResultsBaseline characteristics were balanced. Investigator-assessed PFS was prolonged with alectinib (stratified hazard ratio = 0.43, 95% confidence interval: 0.32–0.58). The median PFS times were 34.8 months with alectinib and 10.9 months with crizotinib. EML4-ALK fusions were detectable in 129 patient plasma samples and 124 tissue samples; variants 1, 2, and 3/ab did not affect PFS, objective response rate, or duration of response. Investigator-assessed PFS was longer for alectinib than for crizotinib across EML4-ALK variants 1, 2, and 3a/b in plasma and tissue. Despite longer treatment duration (27.0 months in the case of alectinib versus 10.8 months in the case of crizotinib), the safety of alectinib compared favorably with that of crizotinib.ConclusionAlectinib continues to demonstrate superior investigator-assessed PFS versus crizotinib in untreated ALK-positive NSCLC, irrespective of EML4-ALK variant.     

Brigatinib Versus Crizotinib in ALK Inhibitor–Naive Advanced ALK-Positive NSCLC: Final Results of Phase 3 ALTA-1L Trial

IntroductionIn the phase 3 study entitled ALK in Lung cancer Trial of brigAtinib in 1st Line (ALTA-1L), which is a study of brigatinib in ALK inhibitor–naive advanced ALK-positive NSCLC, brigatinib exhibited superior progression-free survival (PFS) versus crizotinib in the two planned interim analyses. Here, we report the final efficacy, safety, and exploratory results.MethodsPatients were randomized to brigatinib 180 mg once daily (7-d lead-in at 90 mg once daily) or crizotinib 250 mg twice daily. The primary end point was a blinded independent review committee–assessed PFS. Genetic alterations in plasma cell-free DNA were assessed in relation to clinical efficacy.ResultsA total of 275 patients were enrolled (brigatinib, n = 137; crizotinib, n = 138). At study end, (brigatinib median follow-up = 40.4 mo), the 3-year PFS by blinded independent review committee was 43% (brigatinib) versus 19% (crizotinib; median = 24.0 versus 11.1 mo, hazard ratio [HR] = 0.48, 95% confidence interval [CI]: 0.35–0.66). The median overall survival was not reached in either group (HR = 0.81, 95% CI: 0.53–1.22). Posthoc analyses suggested an overall survival benefit for brigatinib in patients with baseline brain metastases (HR = 0.43, 95% CI: 0.21–0.89). Detectable baseline EML4-ALK fusion variant 3 and TP53 mutation in plasma were associated with poor PFS. Brigatinib exhibited superior efficacy compared with crizotinib regardless of EML4-ALK variant and TP53 mutation. Emerging secondary ALK mutations were rare in patients progressing on brigatinib. No new safety signals were observed.ConclusionsIn the ALTA-1L final analysis, with longer follow-up, brigatinib continued to exhibit superior efficacy and tolerability versus crizotinib in patients with or without poor prognostic biomarkers. The suggested survival benefit with brigatinib in patients with brain metastases warrants future study.     

Establishment of a Conditional Transgenic Mouse Model Recapitulating EML4-ALK–Positive Human Non–Small Cell Lung Cancer

BackgroundAnaplastic lymphoma receptor tyrosine kinase gene (ALK) fusion is a distinct molecular subclassification of NSCLC that is targeted by anaplastic lymphoma kinase (ALK) inhibitors. We established a transgenic mouse model that expresses tumors highly resembling human NSCLC harboring echinoderm microtubule associated protein like 4 gene (EML)-ALK fusion. We aimed to test an EML4-ALK transgenic mouse model as a platform for assessing the efficacy of ALK inhibitors and examining mechanisms of acquired resistance to ALK inhibitors.MethodsTransgenic mouse lines harboring LoxP-STOP-LoxP-FLAGS–tagged human EML4-ALK (variant 1) transgene was established by using C57BL/6N mice. The transgenic mouse model with highly lung-specific, inducible expression of echinoderm microtubule associated protein like 4–ALK fusion protein was established by crossing the EML4-ALK transgenic mice with mice expressing Cre–estrogen receptor fusion protein under the control of surfactant protein C gene (SPC). Expression of EML4-ALK transgene was induced by intraperitoneally injecting mice with tamoxifen. When the lung tumor of the mice treated with the ALK inhibitor crizotinib for 2 weeks was measured, tumor shrinkage was observed.ResultsEML4-ALK tumor developed after 1 week of tamoxifen treatment. Echinoderm microtubule associated protein like 4–ALK was strongly expressed in the lung but not in other organs. ALK and FLAGS expressions were observed by immunohistochemistry. Treatment of EML4-ALK tumor–bearing mice with crizotinib for 2 weeks induced dramatic shrinkage of tumors with no signs of toxicity. Furthermore, prolonged treatment with crizotinib led to acquired resistance in tumors, resulting in regrowth and disease progression. The resistant tumor nodules revealed acquired ALK G1202R mutations.ConclusionsAn EML4-ALK transgenic mouse model for study of drug resistance was successfully established with short duration of tumorigenesis. This model should be a strong preclinical model for testing efficacy of ALK TKIs, providing a useful tool for investigating the mechanisms of acquired resistance and pursuing novel treatment strategies in ALK-positive lung cancer.     

Targeting stemness is an effective strategy to control EML4-ALK+ non-small cell lung cancer cells

The fusion between anaplastic lymphoma kinase (ALK) and echinoderm microtubule-associated protein-like 4 (EML4) is a causative factor in a unique subset of patients with non-small cell lung carcinoma (NSCLC). Although the inhibitor crizotinib, as it blocks the kinase activity of the resulting EML4-ALK fusion protein, displays remarkable initial responses, a fraction of NSCLC cases eventually become resistant to crizotinib by acquiring mutations in the ALK domain or activating bypass pathways via EGFR, KIT, or KRAS. Cancer stem cell (CSC) theory provides a plausible explanation for acquisition of tumorigenesis and resistance. However, the question as to whether EML4-ALK-driven tumorigenesis is linked with the stem-like property and whether the stemness is an effective target in controlling EML4-ALK⁺ NSCLC including crizotinib-resistant NSCLC cells has not been addressed. Here, we report that stem-like properties stem from ALK activity in EML4-ALK⁺ NSCLC cells. Notably, treatment with rapamycin, a CSC targeting agent, attenuates stem-like phenotypes of the EML4-ALK⁺ cells, which increased capability of tumor formation and higher expression of stemness-associated molecules such as ALDH, NANOG, and OCT4. Importantly, combinational treatment with rapamycin and crizotinib leads to synergistic anti-tumor effects on EML4-ALK⁺ NSCLC cells as well as on those resistant to crizotinib. Thus, we provide a proof of principle that targeting stemness would be a novel strategy to control intractable EML4-ALK⁺ NSCLC.     

Sequential Therapy with Crizotinib and Alectinib in ALK-Rearranged Non–Small Cell Lung Cancer—A Multicenter Retrospective Study

IntroductionAlectinib and crizotinib have been approved for the therapy of NSCLC caused by anaplastic lymphoma kinase gene (ALK) rearrangement. The effect of alectinib or crizotinib on overall survival (OS) in patients with ALK-rearranged NSCLC remains unknown.MethodsA multicenter retrospective study was conducted to compare OS between patients receiving alectinib and crizotinib and between patients treated with alectinib and those treated sequentially with crizotinib and then alectinib after crizotinib failure. The time to treatment failure (TTF), progression-free survival (PFS), and OS were compared.ResultsSixty-one patients with ALK-rearranged NSCLC were enrolled. Forty-six patients were treated with anaplastic lymphoma kinase (ALK) inhibitors (31 with crizotinib, 28 with alectinib, and 13 with both ALK inhibitors). The response rate was 66.7% for the crizotinib-treated group and 80.8% for the alectinib-treated group. Among all patients, TTF and PFS were significantly prolonged in the alectinib-treated group compared with in the crizotinib-treated group. Subgroup analyses revealed significantly prolonged TTF for alectinib compared with crizotinib therapy in the ALK inhibitor–naive population. OS was significantly longer in the alectinib-treated group than in the crizotinib-treated group. The TTF and OS of patients treated sequentially with crizotinib and then with alectinib after crizotinib failure tended to be longer than those of patients treated with alectinib alone.ConclusionsTherapy with alectinib alone was significantly superior to therapy with crizotinib alone in terms of TTF, PFS, and OS, and sequential therapy with crizotinib and alectinib after crizotinib failure tended to provide a better OS benefit than did therapy with alectinib alone in patients with ALK-positive NSCLC. However, large-scale prospective studies are needed to confirm these observations.     

Monitoring Therapeutic Response and Resistance: Analysis of Circulating Tumor DNA in Patients With ALK+ Lung Cancer

IntroductionDespite initial effectiveness of ALK receptor tyrosine kinase inhibitors (TKIs) in patients with ALK+ NSCLC, therapeutic resistance will ultimately develop. Serial tracking of genetic alterations detected in circulating tumor DNA (ctDNA) can be an informative strategy to identify response and resistance. This study evaluated the utility of analyzing ctDNA as a function of response to ensartinib, a potent second-generation ALK TKI.MethodsPre-treatment plasma was collected from 76 patients with ALK+ NSCLC who were ALK TKI–naive or had received prior ALK TKI, and analyzed for specific genetic alterations. Longitudinal plasma samples were analyzed from a subset (n = 11) of patients. Analysis of pre-treatment tumor biopsy specimens from 22 patients was compared with plasma.ResultsDisease-associated genetic alterations were detected in 74% (56 of 76) of patients, the most common being EML4-ALK. Concordance of ALK fusion between plasma and tissue was 91% (20 of 22 blood and tissue samples). Twenty-four ALK kinase domain mutations were detected in 15 patients, all had previously received an ALK TKI; G1269A was the most prevalent (4 of 24). Patients with a detectable EML4-ALK variant 1 (V1) fusion had improved response (9 of 17 patients; 53%) to ensartinib compared to patients with EML4-ALK V3 fusion (one of seven patients; 14%). Serial changes in ALK alterations were observed during therapy.ConclusionsClinical utility of ctDNA was shown, both at pre-treatment by identifying a potential subgroup of ALK+ NSCLC patients who may derive more benefit from ensartinib and longitudinally by tracking resistance. Prospective application of this technology may translate to improved outcomes for NSCLC patients treated with ALK TKIs.     

ALK Inhibitors: What Is the Best Way to Treat Patients With ALK+ Non–Small-Cell Lung Cancer?

Genetic insight into the pathogenesis of lung cancer has paved the way for a new era in its treatment. Recently, anaplastic lymphoma kinase (ALK) has been identified as exerting a potent transforming effect through genetic rearrangement in patients with lung cancer. Preclinical and single-arm phase I studies have shown that patients with ALK-rearranged non–small cell lung cancer (NSCLC) can be successfully treated with crizotinib. Furthermore, a phase III randomized study indicated that crizotinib is superior to standard chemotherapy in the treatment of patients with NSCLC harboring the ALK rearrangement who had received 1 previous platinum-based chemotherapy. Despite the excellent efficacy of crizotinib in patients with ALK-positive (ALK⁺) lung cancer, resistance mechanisms—such as secondary mutations in the ALK gene, the activation of other oncogenes, and so on—have been identified as conferring resistance to crizotinib. Second-generation ALK inhibitors, such as alectinib and ceritinib, have been shown to be effective not only in crizotinib-naive patients but also in those resistant to crizotinib. Therefore, although some agents specifically targeting ALK have been developed and their efficacy has been documented, how ALK inhibitors should be administered in the setting of ALK-rearranged NSCLC remains to be fully elucidated. Can second-generation ALK inhibitors replace crizotinib? Is crizotinib just a first-generation ALK inhibitor? Is the sequential use of crizotinib and second-generation ALK inhibitors the best method? In this article, we review the preclinical and clinical results regarding crizotinib and second-generation ALK inhibitors, as well as the resistance mechanisms, and discuss the best methods for treating patients with ALK⁺ NSCLC.     

Clinical outcome for EML4-ALK-positive patients with advanced non-small-cell lung cancer treated with first-line platinum-based chemotherapy

BackgroundThe EML4-ALK fusion oncogene represents a recently identified molecular target in a subset of patients with non-small-cell lung cancer (NSCLC). Limited data have been available, however, on the outcome of first-line platinum-based chemotherapy in patients with EML4-ALK-positive advanced NSCLC who have not been treated with an ALK kinase inhibitor.Patients and methodsThe efficacy of platinum-based chemotherapy was compared between patients with advanced nonsquamous NSCLC who harbor EML4-ALK and those who harbor EGFR mutations and those with neither molecular abnormality.ResultsAmong 200 patients with advanced nonsquamous NSCLC, 18 (9.0%) were positive for EML4-ALK, 31 (15.5%) harbored EGFR mutations, and 151 (75.5%) were wild type for both abnormalities. Platinum-based combination chemotherapy showed similar efficacies in the EML4-ALK, EGFR mutation, and wild-type cohorts in terms of response rate and progression-free survival, and overall survival in the EML4-ALK cohort closely resembled that in the wild-type cohort. Within the EML4-ALK cohort, patients with variants 1 or 3 of the fusion gene were predominant and did not appear to differ in their sensitivity to the platinum-based regimens.ConclusionPatients with EML4-ALK-positive advanced NSCLC manifest an aggressive clinical course similar to that of those with wild-type tumors if the effective targeted therapy is not instituted.     

Identification of a Novel HIP1-ALK Fusion Variant in Non–Small-Cell Lung Cancer (NSCLC) and Discovery of ALK I1171 (I1171N/S) Mutations in Two ALK-Rearranged NSCLC Patients with Resistance to Alectinib

Huntingtin-interacting protein 1 (HIP1) has recently been identified as a new fusion partner fused to anaplastic lymphoma kinase (ALK) in non–small-cell lung cancer (NSCLC). To date, two variants of HIP1-ALK (H21; A20) and (H28; A20) have been identified in NSCLC. However, the response of patients with NSCLC harboring HIP1-ALK to ALK inhibitors and potential resistance mechanisms to such remain unknown. Here, we report a patient with NSCLC harboring a novel HIP1-ALK fusion variant (H30; A20). This patient and another patient with EML4-ALK variant 3a/b initially responded sequentially to crizotinib and then alectinib, a next-generation ALK inhibitor, but developed acquired resistance to alectinib with the presence of a mutation in amino acid residue 1171 (I1171N and I1171S respectively) located in the hydrophobic regulatory spine (R-spine) of the ALK kinase in both the cases as identified by a comprehensive next-generation sequencing-based assay performed on biopsies of new liver metastases that developed during alectinib treatment.     

Sequential Use of Anaplastic Lymphoma Kinase Inhibitors in Japanese Patients With ALK-Rearranged Non–Small-Cell Lung Cancer: A Retrospective Analysis

Background

Second-generation anaplastic lymphoma kinase (ALK) inhibitors, such as alectinib and ceritinib, have recently been approved for treatment of ALK-rearranged non–small-cell lung cancer (NSCLC). An optimal strategy for using 2 or more ALK inhibitors has not been established. We sought to investigate the clinical impact of sequential use of ALK inhibitors on these tumors in clinical practice.

Management of Central Nervous System Metastases in Patients With Advanced Anaplastic Lymphoma Kinase-Rearranged Non–Small-Cell Lung Cancer During Crizotinib Treatment

BackgroundCentral nervous system (CNS) progression is a common manifestation of acquired resistance to crizotinib in anaplastic lymphoma kinase (ALK)-rearranged non–small-cell lung cancer (NSCLC). However, an optimal tailored treatment approach has not been established in patients with CNS failure during crizotinib treatment.Patients and MethodsPatients with ALK-rearranged NSCLC with CNS progression during crizotinib treatment between January 2013 and December 2016 were included for analysis. Clinical data for different treatments after CNS failure during crizotinib treatment were retrospectively collected.ResultsAmong the 44 patients who had CNS progression during crizotinib treatment, 19, 15, 8, and 2 patients received crizotinib treatment beyond progressive disease (CBPD), a second ALK tyrosine kinase inhibitor (TKI), chemotherapy, and best supportive care, respectively. Post progression survival offered by treatment with a second ALK TKI was significantly more favorable than that of chemotherapy (P < .001) or CBPD (P = .045). In addition, patients who received sequential treatment with a second ALK TKI had significantly longer intracranial time to progression (IC-TTP) compared with those treated with chemotherapy (P < .01) or CBPD after radiotherapy (P = .003). In the 7 patients who received brigatinib, the median IC-TTP was 21.8 months (95% confidence interval, 11.7-32.0). The additional use of CNS radiotherapy in patients treated with a second ALK TKI showed no significance in terms of IC-TTP (P = .54).ConclusionAlthough CBPD is an option in patients with isolated CNS progression during crizotinib treatment, sequential treatment with a second ALK TKI, particularly brigatinib, might be preferable. The newly approved TKI, brigatinib, showed promise in the control of brain metastases, even without radiotherapy.     

W‘ALK’ Into the Next Stage

In 2007, the rearrangement of anaplastic lymphoma kinase (ALK) was identified to be associated with the pathogenesis of a subset of patients with non–small-cell lung cancer (NSCLC). Surprisingly, approximately 4 years after the discovery of ALK rearrangement in lung cancer, the first-in-class ALK inhibitor (ALKi), crizotinib, was approved for metastatic ALK-rearranged NSCLC by the US Food and Drug Administration. Subsequently, next-generation ALKis, such as alectinib and ceritinib, have been developed, and some of them have been applied in the clinical setting. Furthermore, various resistance mechanisms against ALKis have been gradually elucidated, and treatment strategies according to such resistance have been proposed. In addition, novel ALKis exhibit good antitumor efficacy for brain metastases. Thus, we now know much about ALK-rearranged NSCLC; however, is it enough? Several concerns, such as the optimal sequence of ALKis, significance of antiangiogenic therapy, immune checkpoint therapy, and cytotoxic chemotherapy in ALK-rearranged NSCLC, should be clearly addressed, which would lead to the establishment of optimal treatment strategies and a more prolonged survival in patients with ALK rearrangement. Thus, we should w‘ALK’ into the next stage.     

Selective ALK inhibitor alectinib with potent antitumor activity in models of crizotinib resistance

The clinical efficacy of the ALK inhibitor crizotinib has been demonstrated in ALK fusion-positive NSCLC; however, resistance to crizotinib certainly occurs through ALK secondary mutations in clinical use. Here we examined the efficacy of a selective ALK inhibitor alectinib/CH5424802 in models of crizotinib resistance. Alectinib led to tumor size reduction in EML4-ALK-positive xenograft tumors that failed to regress fully during the treatment with crizotinib. In addition, alectinib inhibited the growth of some EML4-ALK mutant-driven tumors, including the G1269A model. These results demonstrated that alectinib might provide therapeutic opportunities for crizotinib-treated patients with ALK secondary mutations.     

Anaplastic lymphoma kinase as a new target for the treatment of non-small-cell lung cancer

The anaplastic lymphoma kinase (ALK) gene rearrangement identifies a distinct molecular subset in non-small-cell lung cancer (NSCLC) populations susceptible to targeted inhibition. It consists of a small inversion in the short arm of chromosome 2 between exon 20 of the ALK gene and different exons of the echinoderm microtubule-associated protein-like (EML4) gene. This translocation leads to a chimeric protein with constitutive activation of ALK that possesses an oncogenic activity demonstrated both in vitro and in vivo. Other rare translocation partners for ALK other than EML4 may be found in lung cancers, including TRK-fused gene (TFG) and kinesin family member 5B (KIF5B). ALK-positive patients represent 5–6% of all NSCLCs and they seem to have particular clinicopathological and molecular features. Recently, Phase I–II trial results of crizotinib, a potent dual c-MET and ALK inhibitor, demonstrated its dramatic efficacy in ALK-positive patients with advanced NSCLC. This article will present knowledge on the characteristics of ALK-positive patients, discuss the different methods of ALK rearrangement detection and focus on clinical results of crizotinib.     

Different Types of <i>ROS1</i> Fusion Partners Yield Comparable Efficacy to Crizotinib

ROS1 rearrangements define a distinct molecular subset of non-small-cell lung cancer (NSCLC), which can be treated effectively with crizotinib, a tyrosine kinase inhibitor (TKI) targeting ROS1/MET/ALK rearrangements. Diverse efficacy was observed in ROS1-rearranged NSCLC patients. Because of its rareness, very limited studies have investigated the correlation between different fusion partners and response to crizotinib. In this study, we retrospectively screened 6,235 advanced NSCLC patients (stage IIIB to IV) from five hospitals and identified 106 patients with ROS1 rearrangements based on either plasma or tumor tissue testing using capturebased targeted sequencing. The most frequently occurring fusion partners included cluster of differentiation 74 (CD74), ezrin (EZR), syndecan 4 (SDC4), and tropomyosin 3 (TPM3), occurring in 49.1%, 17%, 14.2%, and 4.7% of patients, respectively. Among them, 38 patients were treated with crizotinib. Seventeen patients were treatment naive, and the remaining were previously treated with pemetrexed-based chemotherapy. Collectively, there was no significant difference among patients with various types of ROS1 fusion partners in overall survival (OS) and progression-free survival (PFS). Patients who were treated with crizotinib as first-line therapy showed comparable PFS (p=0.26) to patients who were previously treated with pemetrexed-based chemotherapy. For treatment-naive patients, patients with low baseline ROS1 allelic fraction (AF) had a statistically significant longer OS than those with high ROS1 AF (184 vs. 110 days, p=0.048). Collectively, our study demonstrates that ROS1⁺ patients with various fusion partners show comparable efficacy to crizotinib.