Second-generation Anaplastic Lymphoma Kinase Research Articles

Alectinib Resistance in ALK-Rearranged Lung Cancer by Dual Salvage Signaling in a Clinically Paired Resistance Model.

The mechanisms responsible for the development of resistance to alectinib, a second-generation anaplastic lymphoma kinase (ALK) inhibitor, are still unclear, and few cell lines are currently available for investigating ALK-rearranged lung cancer. To identify the mechanisms underlying acquired resistance to alectinib, two patient-derived cell lines were established from an alectinib-naïve ALK-rearranged lung cancer and then after development of alectinib resistance. The properties acquired during treatments were detected by comparisons of the two cell lines, and then functional analyses were performed. Coactivation of c-Src and MET was identified after the development of alectinib resistance. Combinatorial therapy against Src and MET significantly restored alectinib sensitivity in vitro (17.2-fold). Increased apoptosis, reduction of tumor volume, and inhibition of MAPK and PI3K/AKT signaling molecules for proliferation and survival were observed when the three kinases (Src, MET, and ALK) were inhibited. A patient-derived xenograft from the alectinib-resistant cells indicated that combination therapy with a saracatinib and crizotinib significantly decreased tumor size in vivo. To confirm the generality, a conventional alectinib-resistant cell line model (H2228-AR1S) was established from NCI-H2228 cells (EML4-ALK variant 3a/b). In H2228-AR1S, combination inhibition of Src and MET also restored alectinib sensitivity. These data reveal that dual salvage signaling from MET and Src is a potential therapeutic target in alectinib-resistant patients. IMPLICATIONS: This study demonstrates the feasibility to elucidate personalized drug-resistance mechanisms from individual patient samples.

Targeted Therapy With Anaplastic Lymphoma Kinase Inhibitor (Alectinib) in Adolescent Metastatic Non–Small Cell Lung Carcinoma

Primary lung adenocarcinoma is extremely rare in the pediatric age group. We report an 18-year-old man with non-small cell lung carcinoma stage IV with brain and bone metastatic. Lung biopsy showed expression of PDL1 along with rearrangement of ALK gene at chromosome 2p23. However, neither mutation of ROS1 nor epidermal growth factor receptor overexpression was seen. Second-generation anaplastic lymphoma kinase (ALK) inhibitor (alectinib) is initiated as first line of treatment. After 8 months of treatment with alectinib, F-NaF PET/CT demonstrated resolution of bone lesions. The present case show rapid and good response to alectinib in metastatic ALK-positive non-small cell lung carcinoma.

Brigatinib in Patients With Alectinib-Refractory ALK-Positive NSCLC.

The second-generation anaplastic lymphoma kinase (ALK) inhibitor alectinib recently showed superior efficacy compared to the first-generation ALK inhibitor crizotinib in advanced ALK-rearranged NSCLC, establishing alectinib as the new standard first-line therapy. Brigatinib, another second-generation ALK inhibitor, has shown substantial activity in patients with crizotinib-refractory ALK-positive NSCLC; however, its activity in the alectinib-refractory setting is unknown. A multicenter, retrospective study was performed at three institutions. Patients were eligible if they had advanced, alectinib-refractory ALK-positive NSCLC and were treated with brigatinib. Medical records were reviewed to determine clinical outcomes. Twenty-two patients were eligible for this study. Confirmed objective responses to brigatinib were observed in 3 of 18 patients (17%) with measurable disease. Nine patients (50%) had stable disease on brigatinib. The median progression-free survival was 4.4 months (95% confidence interval [CI]: 1.8-5.6 months) with a median duration of treatment of 5.7 months (95% CI: 1.8-6.2 months). Among 9 patients in this study who underwent post-alectinib/pre-brigatinib biopsies, 5 had an ALK I1171X or V1180L resistance mutation; ofthese, 1 had a confirmed partial response and 3 had stabledisease on brigatinib. One patient had an ALK G1202R mutation in a post-alectinib/pre-brigatinib biopsy, and had progressive disease as the best overall response to brigatinib. Brigatinib has limited clinical activity in alectinib-refractory ALK-positive NSCLC. Additional studies are needed to establish biomarkers of response to brigatinib and to identify effective therapeutic options for alectinib-resistant ALK-positive NSCLC patients.

A case of ALK-rearranged non-small cell lung cancer that responded to ceritinib after development of resistance to alectinib.

The second-generation anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKIs) alectinib and ceritinib are standard treatment options for patients with non–small cell lung cancer (NSCLC) positive for ALK fusion genes. However, almost all patients eventually develop resistance to these drugs. We here report a case of ALK-rearranged NSCLC that developed resistance to alectinib but remained sensitive to ceritinib. The L1196M mutation within the ALK fusion gene was detected after failure of consecutive treatment with crizotinib and alectinib, but no other mechanism underlying acquired resistance to ALK-TKIs was found to be operative. Given the increasing application of ALK-TKIs to the treatment of patients with ALK-rearranged NSCLC, further clinical evaluation is warranted to provide a better understanding of the mechanisms of acquired resistance to these agents and to inform treatment strategies for such tumors harboring secondary mutations.

The efficacy and safety of alectinib in the treatment of ALK+ NSCLC: a systematic review and meta-analysis.

BackgroundAlectinib is a second-generation anaplastic lymphoma kinase (ALK) inhibitor approved by the US Food and Drug Administration to treat crizotinib-refractory non-small cell lung cancer. We performed this meta-analysis to synthesize the results of different clinical trials to evaluate the efficacy and safety of alectinib.MethodsA search of 3 databases, including PubMed, Web of Science, and the Cochrane Library, was performed from the inception of each database through September 5, 2017. We have pooled the overall response rate (ORR), disease control rate, progression-free survival, and intracranial ORR to evaluate the efficacy of alectinib. Discontinuation rate, rate of dose reduction or interruption due to adverse events as well as the incidence of several adverse events were aggregated to evaluate its safety.ResultsA total of 8 studies with 626 patients have been included in our study. The pooled efficacy parameters are as follows: ORR 70% (95% CI: 57% to 82%), disease control rate 88% (95% CI: 82% to 94%), progression-free survival 9.36 months (95% CI: 7.38% to 11.34%), and intracranial ORR 52% (95% CI: 45% to 59%). ALK inhibitor-naïve patients tend to have better responses than crizotinib-pretreated patients. The aggregate discontinuation rate is 7% (95% CI: 4% to 10%), and the pooled rate of dose reduction or interruption is 33% (95% CI: 24% to 42%). The incidences of most adverse events were relatively low, while the incidences of 2 frequently reported adverse events, myalgia (18%) and anemia (25%), were even higher than with the first-generation ALK inhibitor crizotinib.ConclusionGenerally, alectinib is a drug with preferable efficacy and tolerable adverse effects, and it is suitable for the treatment of intracranial metastases.

Case report: continued treatment with alectinib is possible for patients with lung adenocarcinoma with drug-induced interstitial lung disease

BackgroundAlectinib, a second-generation anaplastic lymphoma kinase (ALK) inhibitor, is a key drug for ALK rearranged lung adenocarcinoma. Interstitial lung disease (ILD) is an important adverse effect of alectinib, which generally requires termination of treatment. However, we treated two patients with drug-induced ILD who continued to receive alectinib.Case presentationPatient 1 was a 57-year-old male with an ALK-rearranged Stage IV lung adenocarcinoma who was administered alectinib as first-line therapy. Computed tomography (CT) detected asymptomatic ground-glass opacity (GGO) on day 33 of treatment. Alectinib therapy was therefore discontinued for 7 days and then restarted. GGO disappeared, and the progression of ILD ceased. Patient 2 was a 64-year-old woman with an ALK-positive lung adenocarcinoma who was administered alectinib as third-line therapy. One year later, CT detected GGO; and she had a slight, nonproductive cough. Alectinib therapy was continued in the absence of other symptoms, and GGO slightly diminished after 7 days. Two months later, CT detected increased GGO, and alectinib therapy was continued. GGO diminished again after 7 days. The patient has taken alectinib for more than 2 years without progression of ILD.ConclusionsCertain patients with alectinib-induced ILD Grade 2 or less may continue alectinib therapy if they are closely managed.

A reliable and stable method for determination of brigatinib in rat plasma by UPLC-MS/MS: Application to a pharmacokinetic study.

Brigatinib is the second-generation anaplastic lymphoma kinase - inhibitor in non-small cell lung cancer and it can overcome the crizotinib-resistance. Chromatographic separation was carried out on an Acquity Ultra Performance Liquid Chromatography (UPLC) unit with a BEH C18 column (2.1mm×50mm, 1.7μm). The mobile phase was composed of acetonitrile and 0.1% formic acid in water. No endogenous interfering compounds was discovered at retention time of brigatinib (0.56min) and imatinib (IS, 1.41min). MS/MS detection was performed in positive mode. And the MRM transitions were m/z 584.09→484.08 and m/z 494.3→394.2 for brigatinib and IS, respectively. This method was assessed to be stable, specificity, and no matrix effect in three concentrations (0.004, 0.4, 4μg/mL). The intra-day and inter-day precisions were less than 11.09% and 6.43%. And intra-day and inter-day accuracies were ranged from -3.88% to 5.44%. The recovery of brigatinib was from 85.26% to 96.05%. Additionally, the method had a good linearity in the range of 0.002-5μg/mL. The presented method was effectively implemented to determine the concentration of brigatinib in rat plasma.

Molecular and clinical features of second-generation anaplastic lymphoma kinase inhibitors: ceritinib.

The discovery of ALK rearrangement in non-small-cell lung cancer (NSCLC) triggered rapid clinical development of a family of specific drugs targeting this alteration, called ALK inhibitors. Despite high rate of responses, the vast majority of patients treated with first-generation ALK inhibitor crizotinib will ultimately develop disease progression. The second-generation ALK inhibitor, ceritinib, is an oral, small-molecule that inhibits the ALK kinase activity with a potency 20-fold greater than crizotinib, being able to tackle some of the principal mechanisms of resistance to crizotinib. Evidences from five large prospective clinical trials have so far showed impressive activity of ceritinib in ALK inhibitor pretreated and naive NSCLC patients. This review will focus on the preclinical and clinical data available regarding ceritinib pharmacology, clinical efficacy and safety profile.

Brigatinib for the treatment of ALK-positive advanced non-small cell lung cancer patients.

Brigatinib (AP-26113, Alunbrig) is a second-generation anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitor (TKI) that is highly active in non-small cell lung cancer (NSCLC) harboring ALK translocation. Brigatinib was found to be very active against different ALK resistance mutations that mediate acquired resistance biology processes, particularly G1269A ALK C1156Y, I1171S/T, V1180L and others. Different clinical trials evaluated the activity of brigatinib in crizotinib-resistant patients, confirming high activity with durable response not only in parenchymal disease, but also in intracranial disease. Nowadays, brigatinib is under evaluation in different clinical trials exploring TKI-naive patients in the first-line setting. On the basis of its significant activity results, brigatinib received approval by the FDA for the treatment of patients with ALK-positive metastatic NSCLC who have progressed on or are intolerant to crizotinib.

Successful ceritinib treatment in a man with MPE and an ALK fusion gene mutation after multiple treatments.

Introduction Ceritinib is a second-generation anaplastic lymphoma kinase (ALK) inhibitor. It inhibits two of the most common ALK-mutants that confer resistance to crizotinib. Ceritinib was approved by Food and Drug Administration in April 2014. However, the efficacy of ceritinib in Asian patients have not been widely studied. Decrease of malignant pleural effusion (MPE) has been rarely reported after treatment with ceritinib.Case descriptionA 50-year old man diagnosed with stage IV lung adenocarcinoma presented with MPE and an ALK fusion gene mutation. The patient showed partial response to ceritinib after 2-month treatment. Ultrasound showed MPE significantly decreased.Discussion and evaluationCeritinib is a good choice, as a targeted therapy, which is more prospect in the advanced cancer patients than the traditional therapy.ConclusionCeritinib seems to have a good efficacy in reducing MPE in advanced Asian lung adenocarcinoma patients, when other chemotherapy failed.

Current evidence in support of the second-generation anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitor alectinib for the treatment of non-small cell lung cancer positive for ALK translocation

Treatment for advanced non-small cell lung cancer (NSCLC) depends on the molecular characteristics of the tumor. Mutations of the epidermal growth factor receptor ( EGFR ) gene are present in ~32% of Asians and ~7% of individuals of other ethnic groups with NSCLC (1), and rearrangements of the anaplastic lymphoma kinase ( ALK ) gene have been detected in ~3% to 5% of NSCLC tumors (2-4). The echinoderm microtubule-associated protein-like 4 ( EML4 ) gene is the most common fusion partner of ALK in NSCLC, and the fusion gene exists in several variants with different breakpoints within EML4 . NSCLC tumors that harbor ALK fusion genes are oncogene addicted and therefore usually sensitive to treatment with ALK tyrosine kinase inhibitors ( ALK -TKIs).

Abstract 2941: Increased expression of ABCB1/MDR1 could be associated with alectinib resistance in ALK-rearranged lung cancer cells

Abstract Alectinib, a second-generation anaplastic lymphoma kinase (ALK) inhibitor, has shown promising activity in ALK-rearranged non-small cell lung cancer (NSCLC). The mechanisms of drug resistance to alectinib have been still unclear. To explore whether overexpression of ATP-binding cassette (ABC) transporters might provide a potential mechanism of alectinib resistance in lung cancer, we established alectinib-resistant cell lines from NCI-H2228 and primary ALK-rearranged cells and evaluated the expression of ABC transporters. We established ALK-rearranged NSCLC cell lines (KTOR 1, 2, and 3) from tumor cells in pleural effusion obtained from 3 patients with alectinib naïve ALK-rearranged NSCLC. In the 2 of 3 patients, tumor cells in pleural effusion were also obtained when disease progression was observed during alectinib treatment (KTOR1-RE and KTOR2-RE). Expression profiling of ABC transporters using quantitative real-time polymerase chain reaction (qRT-PCR) revealed that the expression of ABCB1 was significantly increased in KTOR1-RE and KTOR2-RE compared with the parental cells (4.6 and 10.8 folds), but not other ABC transporters. Then, alectinib-resistant NCI-H2228 cell lines (H2228-AR) were established by exposing the parental cells to stepwise-increasing alectinib up to 5μM for 10 months. We found that the ABCB1/MDR1 expression in H2228-AR cells were significantly increased (2.09-16.6 folds) compared with the parental cells using qRT-PCR and immunoblotting. Then, we examined the alternation of alectinib cytotoxicity by inhibition of ABCB1. Knockdown of ABCB1 expression using small interfering RNA and inhibition of ABCB1 by verapamil enhanced alectinib cytotoxicity in H2228-AR cells. Next, the levels of ABCB1 expression after short-term alectinib exposure were examined in NCI-H2228 and the three alectinib naïve ALK-rearranged NSCLC primary cell lines (KTOR 1, 2, and 3). In all the four ALK-rearranged cell lines, exposure to 100 nM alectinib for 72 hours induced increased expression of ABCB1 compared with control medium. These data suggested that increased expression of ABCB1/MDR1 might be, in part, responsible acquired resistance and sensitivity to alectinib in ALK-rearranged NSCLC cells. Citation Format: Takahiro Tsuji, Hiroaki Ozasa, Yuichi Sakamori, Takashi Nomizo, Yoshitaka Yagi, Hiroki Nagai, Young Hak Kim, Michiaki Mishima. Increased expression of ABCB1/MDR1 could be associated with alectinib resistance in ALK-rearranged lung cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2941.

Abstract 2103: Activating alternative receptor tyrosine kinases induced alectinib-resistance in ALK rearranged non-small cell lung cancer cells

Abstract Background The second-generation anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitor (TKI), alectinib, demonstrated high response rate, long response duration and a favorable toxic profile in patients with ALK-rearranged advanced non-small cell lung cancer in a phase II study (Lancet Oncol 14:590-8, 2013). However, even this promising drug is predicted to develop acquired resistance. Therefore, we investigated the mechanisms of resistance using two alectinib-resistant cell lines. Methods We established alectinib-resistant cell lines, H2228/CHR and ABC-11/CHR, from H2228 (EML4-ALK fusion genes variant 3a/b E6) and ABC-11 (EML4-ALK fusion genes variant 3b E6) respectively, by continuous exposure to alectinib. They were characterized using MTT assay, Western blotting, receptor tyrosine kinase array, ELISA, FISH, RT-PCR, and xenograft models. Results H2228/CHR and ABC-11/CHR cells were 117- and 40-fold more resistant than the parental lines, respectively, and maintained downstream AKT and ERK phosphorylation even in the presence of 10 μM alectinib. There were no ALK secondary mutations in those resistant cell lines. H2228/CHR lost the EML4-ALK fusion gene, and exhibited increased activation of insulin-like growth factor-1 receptor (IGF-1R) and human epidermal growth factor receptor 3 (HER3) with overexpression of the HER3 ligand neuregulin 1. Accordingly, pharmacologic inhibition of IGF-1R and HER3 signaling overcame the resistance. In ABC-11/CHR, MET was activated by stimulated hepatocyte growth factor (HGF) autocrine signaling. We found HGF gene translocation underlying the HGF autocrine system. Anti-HGF antibody suppressed the MET activation and combined treatment with alectinib and anti-HGF antibody or a MET inhibitor suppressed downstream signaling in ABC-11/CHR cells. Finally, crizotinib, which targets both ALK and MET, most effectively inhibited the growth of ABC-11/CHR both in vitro and in vivo. Conclusions We identified novel alectinib resistance mechanisms caused by the activation of alternative tyrosine kinase receptors. Our findings provide new insights into constructing a therapeutic strategy for ALK-positive lung cancer. Citation Format: Hideko Isozaki, Eiki Ichihara, Masayuki Yasugi, Nagio Takigawa, Kadoaki Ohashi, Toshio Kubo, Takashi Ninomiya, Nobuaki Ochi, Daisuke Minami, Kenichiro Kudo, Yuka Kato, Hiroe Kayatani, Tomoki Tamura, Kiichiro Ninomiya, Toshio Higo, Tsuyoshi Makimoto, Akiko Sato, Katsuyuki Hotta, Kunio Matsumoto, Toshiaki Sendo, Mitsune Tanimoto, Katsuyuki Kiura. Activating alternative receptor tyrosine kinases induced alectinib-resistance in ALK rearranged non-small cell lung cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2103.

An Efficient Synthesis of Ceritinib (LDK378) Using a Sandmeyer Reaction

A convenient route for the synthesis of Novartis's second-generation anaplastic lymphoma kinase inhibitor Ceritinib (LDK378) involving starting materials that are commercially available has been achieved. The procedure employed mild reaction conditions and avoided the use of expensive reagents compared to the original synthetic route reported by Novartis. More importantly, gram scale synthesis was accomplished and this protocol could be valid in drug discovery synthesis.

Mechanistic understanding of translational pharmacokinetic-pharmacodynamic relationships in nonclinical tumor models: a case study of orally available novel inhibitors of anaplastic lymphoma kinase.

The orally available novel small molecules PF06463922 [(10R)-7-amino-12-fluoro-2,10,16-trimethyl-15-oxo-10,15,16,17-tetrahydro-2H-8,4-(metheno)pyrazolo[4,3-h][2,5,11]benzoxadiazacyclotetradecine-3-carbonitrile] and PF06471402 [(10R)-7-amino-12-fluoro-2,10,16-trimethyl-15-oxo-10,15,16,17-tetrahydro-2H-8,4-(azeno)pyrazolo[4,3-h][2,5,11]benzoxadiazacyclo-tetradecine-3-carbonitrile] are second-generation anaplastic lymphoma kinase (ALK) inhibitors targeted to both naïve and resistant patients with non-small cell lung cancer (NSCLC) to the first-generation ALK inhibitor crizotinib. The objectives of the present study were to characterize and compare the pharmacokinetic-pharmacodynamic (PKPD) relationships of PF06463922 and PF06471402 for target modulation in tumor and antitumor efficacy in athymic mice implanted with H3122 NSCLC cells expressing a crizotinib-resistant echinoderm microtubule-associated protein-like 4 (EML4)-ALK mutation, EML4-ALK(L1196M). Furthermore, the PKPD relationships for these ALK inhibitors were evaluated and compared between oral administration and subcutaneous constant infusion (i.e., between different pharmacokinetic [PK] profiles). Oral and subcutaneous PK profiles of these ALK inhibitors were adequately described by a one-compartment PK model. An indirect response model extended with a modulator fit the time courses of PF06463922- and PF06471402-mediated target modulation (i.e., ALK phosphorylation) with an estimated unbound EC50,in vivo of 36 and 20 nM, respectively, for oral administration, and 100 and 69 nM, respectively, for subcutaneous infusion. A drug-disease model based on the turnover concept fit tumor growth curves inhibited by PF06463922 and PF06471402 with estimated unbound tumor stasis concentrations of 51 and 27 nM, respectively, for oral administration, and 116 and 70 nM, respectively, for subcutaneous infusion. Thus, the EC50,in vivo to EC60,in vivo estimates for ALK inhibition corresponded to the concentrations required tumor stasis in all cases, suggesting that the pharmacodynamic relationships of target modulation to antitumor efficacy were consistent among the ALK inhibitors, even when the PK profiles with different administration routes were considerably different.

ASCO 2013: new developments in lung cancer

At American Society of Clinical Oncology meeting 2013, some new data were presented, which might have a practice-changing potential. Epidermal growth factor receptor mutational testing was successfully performed in plasma samples, giving hope that tumor biopsies might be replaced by a more comfortable blood analysis. Some studies focused on new second-generation anaplastic lymphoma kinase inhibitors, which showed an impressive efficacy even in crizotinib refractory patients in phase I/II studies and support a sequence of tyrosine kinase inhibitors (TKIs) in this sub-entity of non-small-cell lung cancer (NSCLC). Immunological strategies are worth being tested also in lung cancer, as different strategies have proven first signs of efficacy (MUC1 antigen-specific cancer immunotherapy, programmed cell death ligand 1). Finally, a large phase III trial found that a new oral vascular endothelial growth factor receptor, fibroblast growth factor receptor, and platelet-derived growth factor receptor TKI (nintedanib) combined with docetaxel improved outcome in second-line NSCLC patients when compared with docetaxel monotherapy.