Heterogeneity and Regulatory Mechanisms of ALK-Mutant and Wild-Type Epithelial Cells in Non-Small-Cell Lung Cancer: Single-Cell Transcriptomics and Chromatin Accessibility

Ensartinib as Treatment for ALK-Rearranged NSCLC

Molecular Testing for Stage IV Non-Small Cell Lung Cancer Patients With Targetable Mutations Following Disease Progression.

Non-small cell lung cancers (NSCLCs) harboring ALK gene rearrangements (ALK+) typically become resistant to the first-generation anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitor (TKI) crizotinib through development of secondary resistance mutations in ALK or disease progression in the brain. Mutations that confer resistance to second-generation ALK TKIs ceritinib and alectinib have also been identified. Here, we report the structure and first comprehensive preclinical evaluation of the next-generation ALK TKI brigatinib. A kinase screen was performed to evaluate the selectivity profile of brigatinib. The cellular and in vivo activities of ALK TKIs were compared using engineered and cancer-derived cell lines. The brigatinib-ALK co-structure was determined. Brigatinib potently inhibits ALK and ROS1, with a high degree of selectivity over more than 250 kinases. Across a panel of ALK+ cell lines, brigatinib inhibited native ALK (IC50, 10 nmol/L) with 12-fold greater potency than crizotinib. Superior efficacy of brigatinib was also observed in mice with ALK+ tumors implanted subcutaneously or intracranially. Brigatinib maintained substantial activity against all 17 secondary ALK mutants tested in cellular assays and exhibited a superior inhibitory profile compared with crizotinib, ceritinib, and alectinib at clinically achievable concentrations. Brigatinib was the only TKI to maintain substantial activity against the most recalcitrant ALK resistance mutation, G1202R. The unique, potent, and pan-ALK mutant activity of brigatinib could be rationalized by structural analyses. Brigatinib is a highly potent and selective ALK inhibitor. These findings provide the molecular basis for the promising activity being observed in ALK+, crizotinib-resistant patients with NSCLC being treated with brigatinib in clinical trials. Clin Cancer Res; 22(22); 5527-38. ©2016 AACR.

<div>Abstract<p><b>Purpose:</b> Non–small cell lung cancers (NSCLCs) harboring <i>ALK</i> gene rearrangements (ALK<sup>+</sup>) typically become resistant to the first-generation anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitor (TKI) crizotinib through development of secondary resistance mutations in ALK or disease progression in the brain. Mutations that confer resistance to second-generation ALK TKIs ceritinib and alectinib have also been identified. Here, we report the structure and first comprehensive preclinical evaluation of the next-generation ALK TKI brigatinib.</p><p><b>Experimental Design:</b> A kinase screen was performed to evaluate the selectivity profile of brigatinib. The cellular and <i>in vivo</i> activities of ALK TKIs were compared using engineered and cancer-derived cell lines. The brigatinib–ALK co-structure was determined.</p><p><b>Results:</b> Brigatinib potently inhibits ALK and ROS1, with a high degree of selectivity over more than 250 kinases. Across a panel of ALK<sup>+</sup> cell lines, brigatinib inhibited native ALK (IC<sub>50</sub>, 10 nmol/L) with 12-fold greater potency than crizotinib. Superior efficacy of brigatinib was also observed in mice with ALK<sup>+</sup> tumors implanted subcutaneously or intracranially. Brigatinib maintained substantial activity against all 17 secondary ALK mutants tested in cellular assays and exhibited a superior inhibitory profile compared with crizotinib, ceritinib, and alectinib at clinically achievable concentrations. Brigatinib was the only TKI to maintain substantial activity against the most recalcitrant ALK resistance mutation, G1202R. The unique, potent, and pan-ALK mutant activity of brigatinib could be rationalized by structural analyses.</p><p><b>Conclusions:</b> Brigatinib is a highly potent and selective ALK inhibitor. These findings provide the molecular basis for the promising activity being observed in ALK<sup>+</sup>, crizotinib-resistant patients with NSCLC being treated with brigatinib in clinical trials. <i>Clin Cancer Res; 22(22); 5527–38. ©2016 AACR</i>.</p></div>

<div>Abstract<p><b>Purpose:</b> Non–small cell lung cancers (NSCLCs) harboring <i>ALK</i> gene rearrangements (ALK<sup>+</sup>) typically become resistant to the first-generation anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitor (TKI) crizotinib through development of secondary resistance mutations in ALK or disease progression in the brain. Mutations that confer resistance to second-generation ALK TKIs ceritinib and alectinib have also been identified. Here, we report the structure and first comprehensive preclinical evaluation of the next-generation ALK TKI brigatinib.</p><p><b>Experimental Design:</b> A kinase screen was performed to evaluate the selectivity profile of brigatinib. The cellular and <i>in vivo</i> activities of ALK TKIs were compared using engineered and cancer-derived cell lines. The brigatinib–ALK co-structure was determined.</p><p><b>Results:</b> Brigatinib potently inhibits ALK and ROS1, with a high degree of selectivity over more than 250 kinases. Across a panel of ALK<sup>+</sup> cell lines, brigatinib inhibited native ALK (IC<sub>50</sub>, 10 nmol/L) with 12-fold greater potency than crizotinib. Superior efficacy of brigatinib was also observed in mice with ALK<sup>+</sup> tumors implanted subcutaneously or intracranially. Brigatinib maintained substantial activity against all 17 secondary ALK mutants tested in cellular assays and exhibited a superior inhibitory profile compared with crizotinib, ceritinib, and alectinib at clinically achievable concentrations. Brigatinib was the only TKI to maintain substantial activity against the most recalcitrant ALK resistance mutation, G1202R. The unique, potent, and pan-ALK mutant activity of brigatinib could be rationalized by structural analyses.</p><p><b>Conclusions:</b> Brigatinib is a highly potent and selective ALK inhibitor. These findings provide the molecular basis for the promising activity being observed in ALK<sup>+</sup>, crizotinib-resistant patients with NSCLC being treated with brigatinib in clinical trials. <i>Clin Cancer Res; 22(22); 5527–38. ©2016 AACR</i>.</p></div>

Despite the development and approval of seven anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKIs) spanning over three "generations" since the discovery of ALK fusion positive (ALK+) non-small cell lung cancer (NSCLC), there remains intrinsic and acquired resistances to these approved TKIs. Currently, a fourth-generation (4G) ALK TKI, NVL-655, is being developed to attack some of the unmet needs such as compound resistance mutations in cis. However, EML4-ALK variant 3 and TP53 mutations are intrinsic genomic alterations that negatively modulate efficacy of ALK TKIs. Potentially, in the shifting landscape where lorlatinib should be the first-line ALK TKI of choice based on the CROWN trial, the central β-sheet #6 (Cβ6) mutation ALK L1256F will be the potential acquired resistance mutation to lorlatinib which may be resistant to current ALK TKIs. Here we opine on what additional capacities a putative fifth-generation (5G) ALK TKI will need to possess if it can be achieved in one single molecule. We propose randomized trial schemas targeting some of the intrinsic resistance mechanisms that will lead to approval of a prototypic fifth-generation (5G) ALK TKI and actually be beneficial to ALK+ NSCLC patients rather than just design a positive pivotal superiority trial for the sole purpose of drug approval.

Next-generation Sequencing Identified a Novel WDPCP-ALK Fusion Sensitive to Crizotinib in Lung Adenocarcinoma

ALK rearrangements predict for sensitivity to ALK tyrosine kinase inhibitors (TKIs). However, responses to ALK TKIs are generally short-lived. Serial molecular analysis is an informative strategy for identifying genetic mediators of resistance. Although multiple studies support the clinical benefits of repeat tissue sampling, the clinical utility of longitudinal circulating tumor DNA analysis has not been established in ALK-positive lung cancer. Using a 566-gene hybrid-capture next-generation sequencing (NGS) assay, we performed longitudinal analysis of plasma specimens from 22 ALK-positive patients with acquired resistance to ALK TKIs to track the evolution of resistance during treatment. To determine tissue-plasma concordance, we compared plasma findings to results of repeat biopsies. At progression, we detected an ALK fusion in plasma from 19 (86%) of 22 patients, and identified ALK resistance mutations in plasma specimens from 11 (50%) patients. There was 100% agreement between tissue- and plasma-detected ALK fusions. Among 16 cases where contemporaneous plasma and tissue specimens were available, we observed 100% concordance between ALK mutation calls. ALK mutations emerged and disappeared during treatment with sequential ALK TKIs, suggesting that plasma mutation profiles were dependent on the specific TKI administered. ALK G1202R, the most frequent plasma mutation detected after progression on a second-generation TKI, was consistently suppressed during treatment with lorlatinib. Plasma genotyping by NGS is an effective method for detecting ALK fusions and ALK mutations in patients progressing on ALK TKIs. The correlation between plasma ALK mutations and response to distinct ALK TKIs highlights the potential for plasma analysis to guide selection of ALK-directed therapies.

SY14-3 - Treatment of EML4-ALK Rearranged NSCLC

The anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitor (TKI) alectinib was approved in Japan in 2014 for the treatment of ALK fusion gene-positive advanced non-small cell lung cancer (NSCLC). With the approvals of crizotinib in 2012 and ceritinib in 2017, Japan became the first country with multiple ALK TKIs available for first-line or later use in patients with ALK-positive advanced NSCLC. Here, we collected and evaluated real-world data on ALK TKI clinical usage patterns and sequencing in patients with ALK-positive NSCLC in Japan. This retrospective observational study used the Japanese Medical Data Vision database to analyze data from patients with a confirmed diagnosis of lung cancer who visited a healthcare facility in the database between April 2010 and March 2017, underwent an ALK test, received a prescription for an ALK TKI, and were at least 18years old as of the date of the first ALK TKI prescription. There were no exclusion criteria. Descriptive analyses of demographics, baseline characteristics, ALK TKI treatment patterns and sequences, non-ALK TKI treatments received before, during, and after ALK TKI treatment, and treatment durations were reported. A total of 378 patients met the inclusion criteria and were evaluated in mutually exclusive groups of patients receiving one, two, or three ALK TKIs. The initial ALK TKI prescribed was crizotinib for 52.1% of patients and alectinib for 47.9% of patients; however, the proportion of patients receiving alectinib as the initial ALK TKI increased over time following the Japanese approval of alectinib in 2014. Of the 117 patients who received two or three ALK TKIs, 106 received crizotinib as the first ALK TKI and 11 received alectinib. Before the date of the patient's first ALK TKI prescription, 153 of 378 patients (40.5%) had received chemotherapy. Of 104 patients who discontinued ALK therapy, 46.2% received chemotherapy and 5.8% received immunotherapy as their next treatment. At the time of this analysis, most patients who received more than one ALK TKI received crizotinib as the initial ALK TKI. Additional ALK TKIs have since been approved in Japan as first-line or later therapeutic options for patients with ALK-positive NSCLC, but the optimal sequence of ALK TKI usage remains undetermined. As new data continue to emerge, additional research will be warranted to evaluate ALK TKI sequences that do not include crizotinib as the first therapy in this patient population.

Background: Patients with advanced non-small cell lung cancer (NSCLC) have considerably benefited from the molecular identification of epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) mutations and subsequent targeted therapy against these biomarkers. Few studies have been undertaken in the Indian population on the analysis of both EGFR and ALK mutations in lung adenocarcinoma cases. Aim and Objectives: The aim of this study was to determine the prevalence of EGFR and ALK mutations in lung adenocarcinoma patients, as well as to link mutational status with age, sex, and smoking history. Materials and Methods: This single hospital-based retrospective study was conducted at the Department of Medical Oncology, Acharya Harihar Post Graduate Institute of Cancer, Cuttack, on histologically proven lung adenocarcinoma cases over a duration of two years from 01.08.2019 to 31.07.2021. Results: Out of a total of 164 cases, males comprised 89 (54.26%) of the 164 lung adenocarcinoma cases, while females comprised 75 (45.73%). EGFR mutations were found in 42 (26.75%) of the patients. In 9 cases, the ALK gene rearrangement was also determined to be positive (5.66%). In terms of EGFR and ALK mutations, there was no statistically significant relationship between patient age and gender. (P-value < 0.05). In our research, we found a link between nonsmokers and EGFR and ALK mutations. (P-value <0.05). The deletion of exon 19 (76.19%) was the most prevalent mutation, followed by the exon 21 L858R mutation (14.28%). Conclusion: This study was found to have a significantly higher rate of EGFR and ALK mutation in the Indian population with adenocarcinoma of lung compared to Western populations. To get the maximum benefit from targeted therapies, all patients of adenocarcinoma of the lung should have mutational testing for EGFR and ALK as part of a broad molecular pannel.

9027 Background: NG ALK TKIs are the standard first-line therapy for patients with advanced ALK + NSCLC. Brigatinib has activity against ALK resistance mutations and has CNS activity. The efficacy of brigatinib after NG ALK TKI is undetermined. Methods: Pts with stage IIIB/IV ALK + NSCLC, and PD after NG ALK TKI’s were eligible. Pts were required to have measurable disease, performance status 0-2, and adequate organ function. Pts were required to undergo tumor biopsy ≤ 60 days of enrollment; ctDNA was collected at baseline, at time of imaging, and PD. Brigatinib treatment was 90 mg daily for 7 days and then 180 mg daily (1 cycle = 28 days). Primary objective was objective response rate (ORR), and ORR of 20% was determined to be worthy of further investigation. Response was assessed every 2 cycles. A 2-stage design was used; if > 2 responses in 20 pts in the 1st stage, study proceeded to 2nd stage; if ≥ 5 responses in 40 pts therapy considered active. Results: Between 3/2017 and 11/2018, 20 pts were enrolled in the 1st stage. Pt characteristics were: median age 55 years (range 32 to 71), median number of prior therapies 3 (range 1 to 6), 12 had CNS disease at the baseline (8 pts had CNS PD). ALK resistance mutation detected in 3 of 8 pts on standard of care molecular testing. ORR results were confirmed partial response (n = 8), stable disease (n = 7), PD (n = 3), unconfirmed response (n = 1), and non-evaluable due adverse events (AE’s) (n = 1). The grade 3 or 4 AE’s were: pneumonitis (n = 2); 1 of each: acute renal failure, respiratory failure, sepsis, hypertension, CPK elevation, and headache. With median follow-up of 6.7 months (11 PFS events), the median PFS was 6.4 months (95% CI: 4.6 to NE). Overall survival data is immature. 3 of 3 pts with a known ALK mutation (F1174C in 2 pts, I1171T in 1 pt) responded, 2 of 5 pts without ALK mutation responded. Additional study related tumor analysis and ctDNA analyses are ongoing. Conclusions: Brigatinib has activity after progression on next generation ALK TKI. The study is enrolling additional patients in the second stage. Detailed molecular and site of disease (CNS versus extra-CNS) activity analyses are underway Clinical trial information: NCT02706626.

Next-Generation Sequencing Identified a Novel Crizotinib-Sensitive PLB1-ALK Rearrangement in Lung Large-Cell Neuroendocrine Carcinoma

Lung cancer has opened a new era in cancer treatment by elucidating the tumor's molecular structure and identifying the targetable mutations. Identifying the targeted mutations in lung cancer constitutes one of the main steps of treatment planning. The frequency of EGFR (epidermal growth factor receptor gene) and ALK (anaplastic lymphoma kinase gene) mutations in non-small cell lung cancer (NSCLC) also varies in populations depending on ethnicity, gender, smoking, and histopathological subtype. In general, limited data are available regarding the frequency and regional distribution of these mutations in the Turkish population. Our study aimed to determine the frequency of EGFR and ALK mutations in patients with advanced-stage NSCLC and compare the clinical characteristics, treatment, and survival results of cases with mutations with the group without mutations. In our study, 593 patients with advanced-stage NSCLC diagnosis and mutational analyses were evaluated retrospectively. Demographic characteristics, tumor stages (tumor, node, metastasis, TNM), EGFR and ALK analysis results, treatments applied, and survival of the cases were recorded. EGFR analysis, exon 18, 19, 20, and 21 mutations were studied with real-time PCR (RT-PCR) Rotor-Gene system from patients' samples. For ALK analysis, the ALK Break Apart kit (Zytovision GmbH; Germany) was used with the fluorescent in situ hybridization (FISH) method. In our study, EGFR mutation was detected in 63 patients (10.6%) and ALK mutation in 19 patients (3.2%) out of 593 patients. EGFR mutation was observed more frequently in women and non-smokers (P = 0.001, P = 0.003). No correlation was found between the presence of EGFR mutation and metastases regions and recurrence (P > 0.05). ALK mutation was observed more frequently in non-smokers and females (P = 0.001, P = 0.003). Patients with ALK mutations were younger than other groups (P = 0.003). There was also no significant relationship between ALK mutation and metastates regions and recurrence after treatment (P > 0.05). Patients with EGFR or ALK mutations had a longer life span than other cases (P = 0.474). Those who had ALK mutations and received targeted therapy had a longer average life expectancy (P < 0.05). No difference was observed in those who had EGFR mutations and received targeted treatment in terms of survival (P > 0.05). In our study, conducted in the Aegean region of Turkey, the positivity rates of EGFR and ALK mutations were found to be at similar rates with the Caucasian race across the world. EGFR mutation was more common in women, non-smokers, and patients with adenocarcinoma histology. ALK mutation was also detected more frequently in younger patients, women, and non-smokers. Patients with EGFR and ALK mutations had a longer life expectancy than those without the mutation. It was observed that testing patients diagnosed with advanced-stage NSCLC for genetic mutations of the tumor in the first step of the treatment and initiating treatment in patients with mutations provided a significant survival advantage.

Introduction Anaplastic lymphoma kinase (ALK) gene rearrangement is detected in approximately 3–5% of non-small cell lung cancer (NSCLC) cases. Tyrosine kinase inhibitors (TKIs) targeting ALK rearrangement (ALK–TKIs) have shown significant efficacy and improved the survival of patients with NSCLC exhibiting ALK rearrangement. However, almost all patients exhibit disease progression during TKI therapy owing to resistance acquired through various molecular mechanisms, including both ALK-dependent and ALK-independent. Areas covered Here, we review the mechanisms underlying resistance to second-generation ALK–TKIs, and the clinical management strategies following resistance in patients with ALK rearrangement-positive NSCLC. Expert opinion Treatment strategies following the failure of second-generation ALK–TKIs failure should be based on resistant mechanisms. For patients with ALK mutations who exhibit resistance to second-generation ALK–TKIs, lorlatinib is the primary treatment option. However, the identification of resistance profiles of second-generation ALK–TKIs can aid in the selection of an appropriate treatment strategy. In cases of ALK-dependent resistance mutations, lorlatinib could be the first choice as it exhibits the broadest coverage of mutations that lead to resistance against second-generation ALK–TKIs, such as G1202R, and L1196M. In cases of no resistance mutations, atezolizumab, bevacizumab, and platinum-based chemotherapy could be the alternative treatment options.