Search

Mutations in the ALK gene are detectable in approximately 40% of ALK-rearranged lung cancers resistant to ALK inhibitors. Although epithelial-to-mesenchymal transition (EMT) is a mechanism of resistance to various targeted drugs, its involvement in ALK inhibitor resistance is largely unknown. In this study, we report that both ALK-mutant L1196M and EMT were concomitantly detected in a single crizotinib-resistant lesion in a patient with ALK-rearranged lung cancer. Digital PCR analyses combined with microdissection after IHC staining for EMT markers revealed that ALK L1196M was predominantly detected in epithelial-type tumor cells, indicating that mesenchymal phenotype and ALK mutation can coexist as independent mechanisms underlying ALK inhibitor–resistant cancers. Preclinical experiments with crizotinib-resistant lung cancer cells showed that EMT associated with decreased expression of miR-200c and increased expression of ZEB1 caused cross-resistance to new-generation ALK inhibitors alectinib, ceritinib, and lorlatinib. Pretreatment with the histone deacetylase (HDAC) inhibitor quisinostat overcame this resistance by reverting EMT in vitro and in vivo. These findings indicate that HDAC inhibitor pretreatment followed by a new ALK inhibitor may be useful to circumvent resistance constituted by coexistence of resistance mutations and EMT in the heterogeneous tumor. Significance: These findings show that dual inhibition of HDAC and ALK receptor tyrosine kinase activities provides a means to circumvent crizotinib resistance in lung cancer.

A 51-year-old, male, non-smoker with a 3.4 cm mass in the right middle lobe was diagnosed with large cell neuroendocrine carcinoma (LCNEC). Fluorescence in situ hybridization revealed anaplastic lymphoma kinase (ALK) gene translocation, in agreement with the immunohistochemistry result obtained with use of ALK-Ventana. Radiographic examinations showed both bone and brain metastasis. After two cycles of chemotherapy consisting of etoposide and cisplatin, the patient achieved stable disease, and was subsequently switched to crizotinib. Both computed tomography and magnetic resonance imaging revealed partial response after 4 months of crizotinib, but progressed after treatment for 10 months, when several hard lymph nodes were palpable in the left supraclavicular fossa. Lymph node biopsy showed similar histology of tumor cells and targeted next-generation sequencing revealed ALK F1174L on exon 23 with two rare forms of ALK rearrangements. This case provides evidence of responsiveness of ALK inhibitors for a rare pattern of ALK-rearranged LCNEC, and suggests that F1174L, a common resistant mutation found in non-small-cell lung cancer, also causes crizotinib resistance in LCNEC.

An inflammatory myofibroblastic tumor (IMT) is a rare mesenchymal neoplasm that typically develops in the lungs and seldom in the head and neck region. It is often related to the anaplastic lymphoma kinase (ALK) fusion gene. Crizotinib, a first-generation ALK inhibitor, has been shown to have a notable response in patients with ALK-positive IMT. Here, we report the first case of a 46-year-old man with IMT harboring a novel SQSTM1-ALK fusion gene who demonstrated marked response to alectinib. The patient presented a right neck mass (5-cm diameter) that progressively enlarged and expanded to the upper mediastinum. ALK-rearranged IMT was diagnosed after complete tumor resection. Spindle cells displayed diffuse cytoplasmic staining for ALK on immunohistochemistry. A fluorescence in situ hybridization analysis revealed the translocation of a part of the ALK gene locus at chromosome 2p23. FoundationOne CDx™ assay identified an SQSTM1-ALK gene fusion. After a year, right cervical, subclavian, and mediastinal lymph node metastases, considered unresectable, developed. Notably, the patient exhibited a marked response to alectinib treatment and has sustained for 17 months following systemic therapy initiation without significant adverse events. This report highlights the possibility of alectinib being a reasonable option for advanced IMT with the SQSTM1-ALK fusion.

Our effort toward the process improvement of anaplastic lymphoma kinase (ALK) inhibitor ASP3026 (1) is described. A cost-effective and practical synthesis of 1 was accomplished as a result of the change of starting material from 2,4-dichloro-1,3,5-triazine (6) to cyanuric chloride (9) and late-stage introduction of a highly reactive N-methyl piperazine moiety by reductive amination of intermediate ketone 13. The modified process avoided the challenges with the original synthesis and furnished the several hundred kilograms of high-quality API with high economic efficiency, operability, and reproducibility. Furthermore, a sequence of investigation of polymorphic control in the second-generation synthetic route to obtain the thermodynamically desired, most stable polymorph Form A04 is also discussed.

The objective of this study was to understand outcomes of patients treated with ALK inhibitors, especially when ALK inhibitors are followed by other ALK inhibitors. A systematic literature review was conducted in PubMed, Embase, and Cochrane through July 17, 2017. Conference abstracts (three meetings in past 2 years) also were searched. Of 504 unique publications, 80 met inclusion criteria (47 clinical trials, 33 observational studies). Observational studies have the potential to provide information for ALK inhibitors used sequentially. Ten observational studies reported median overall survival of crizotinib-led sequences ranging from 30.3 to 63.75 months from initiation of crizotinib; 49.4-89.6 months from metastatic non-small-cell lung cancer diagnosis; and 15.5-22.0 months from initiation of the second-generation ALK inhibitor after initial crizotinib. Sequencing of ALK inhibitors may benefit patients progressing on initial ALK inhibitors.

With the advent of next-generation sequencing (NGS) and precision medicine, investigators have determined that tumors from different tissue sources that have the same types of genetic mutations will have a positive response to the same targeted therapy. This finding has prompted us to seek potential therapeutic targets for patients with carcinoma of unknown primary (CUP) using NGS technology. Here, we reported a case of a woman with CUP resistance to chemotherapy. We detected 450 cancer-related gene alterations using three metastatic tumor specimens and found the presence of EML4 exon13 and ALK exon20 fusion. The tumor did respond to crizotinib, a first-generation ALK inhibitor. When her tumor progressed, circulating tumor DNA detection revealed ALK L1196 M and G1269A mutation resistance to crizotinib, but she had a response to brigatinib. This case revealed that NGS technology used to detect the genetic alterations in patients with CUP might be a reliable method to find potential therapeutic targets, although the primary lesion could not always be confirmed. Key Points This case exemplifies responsiveness to ALK inhibitor in carcinoma of unknown primary (CUP) with EML4-ALK fusion. Next-generation sequencing is an important diagnostic tool to find potential therapeutic targets in CUP. Liquid biopsy may be useful to provide critical information about resistance mechanisms in CUP to guide sequential treatment decision with targeted therapy.

Objective: We review here the pharmacology, pharmacokinetics, efficacy, safety, dosage and administration, potential drug-drug interactions and place in therapy of brigatinib for abnormal anaplastic lymphoma kinase (ALK) specific non–small-cell lung cancer (NSCLC). Data Sources: A literature search using PubMed was conducted using the terms brigatinib and ALK positive NSCLC from January 2013 to November 2018. Study Selection and Data Extraction: All English-language articles evaluating brigatinib were analyzed for this review. Data Synthesis: Brigatinib was granted approval for the treatment of patients with metastatic ALK+ NSCLC who have progressed on or are intolerant to crizotinib. It is administered at a dose of 90 mg orally once daily for the first 7 days then, if tolerated, increased to a dose of 180 mg orally once daily. Common adverse effects include nausea, fatigue, diarrhea, increased creatine phosphokinase levels, headache, dyspnea, and hypertension. Serious treatment-emergent adverse effects were pulmonary related. Relevance to Patient Care and Clinical Practice: This article discusses the clinical trials that led to the accelerated approval of brigatinib for its ability to overcome crizotinib-resistant mutations and for its increased central nervous system penetration properties. Conclusion: Brigatinib was granted accelerated approval for the treatment of patients with metastatic ALK+ NSCLC who have progressed on or are intolerant to crizotinib. In a subset of NSCLC patients, brigatinib increases survival for approximately 1 year; however, side effects were detected.

Objective Crizotinib has demonstrated promising efficacy in patients with anaplastic lymphoma kinase (ALK)-positive non-small-cell lung cancer (NSCLC) in clinical trials. We conducted this retrospective multicenter study to assess the outcomes of crizotinib therapy in, to our knowledge, a large sample cohort of patients with ALK-positive advanced NSCLC. Methods We reviewed the medical records of 484 unselected ALK-positive NSCLC patients treated with crizotinib at 5 cancer centers in China from January 2013 to November 2017. Clinical data were collected from the initiation of crizotinib therapy to Response Evaluation Criteria in Solid Tumors (RECIST)-defined progressive disease (PD). Results A total of 428 eligible ALK-positive NSCLC patients were enrolled, 273 (63.8%) of whom received crizotinib as first-line treatment. The median progression-free survival (PFS) and overall survival (OS) from the initiation of crizotinib treatment were 14.4 [95% confidence interval (95% CI), 12.4−16.4] months and 53.4 (95% CI, 33.7−73.1) months, respectively. In subgroup analyses, patients who received crizotinib as first-line treatment showed a higher disease control rate (DCR) and a longer median OS compared with second-/later-line crizotinib treatment (94.8% and OS not reachedvs. 89.0% and 40.5 months, respectively). For 261 patients with RECIST-defined PD, multivariate Cox analysis revealed that in patients who received first-line crizotinib therapy, continued crizotinib beyond progressive disease (CBPD) and next-generation ALK inhibitors after crizotinib failure were associated with improved survival. Conclusions This study has demonstrated the clinically meaningful benefit of crizotinib treatment in a large cohort of Chinese ALK-positive NSCLC patients. CBPD and next-generation ALK inhibitor treatment may provide improved survival after RECIST-defined progression on crizotinib.

Background Patients with adenocarcinoma of the lung are routinely screened for anaplastic lymphoma kinase (ALK) rearrangement because they can be treated by ALK-specific targeted therapy. The clinical and molecular characteristics of large-cell neuroendocrine carcinoma (LCNEC) associated with ALK rearrangement are still unclear. Herein, we assessed the ALK status in a series of patients with LCNEC by testing methods commonly used for adenocarcinoma. Materials and methods ALK expression was first examined by immunohistochemistry. For a positively stained tumor, molecular analyses were then conducted. The ALK fusion partner found in a patient with ALK rearrangement was further identified by direct DNA sequencing. Patient clinicopathological features were also analyzed, focusing on the ALK rearrangement-positive case. Results Immunohistochemistry of seven patients identified strong ALK expression in one case of stage IV LCNEC. Molecular analysis identified a novel rearranged gene resulting from the fusion of kinesin family member 5B (KIF5B) exon 17 to ALK exon 20. The patient was treated with ALK-specific inhibitors, crizotinib and later, alectinib, and has remained alive for more than 24 months without disease progression. Three of the remaining six patients without ALK rearrangement had stage IV cancer and received cytotoxic chemotherapies. Their average overall survival was 5.4 months. Conclusion To our knowledge, this is the first report of a KIF5B-ALK fusion gene in LCNEC. The patient was successfully treated with ALK inhibitors, suggesting that sensitivity to ALK inhibitor may define a specific LCNEC subtype. We propose that screening for ALK rearrangement in patients with LCNEC may assist in selecting potential candidates for targeted therapy.

Aiming to develop promising ALK inhibitors, two series of N2-(2-alkyoxy-6-aliphatic aminopyridin-3-yl)-2,4-diaminepyrimidine derivatives (22a-x and 23a-d) were designed according to scaffold hopping and bioisosterism principles. All compounds were efficiently synthesized by concise reactions and anti-proliferative activities on ALK-addicted H2228, Karpas299 cells and EGFR-expressive A549 cell were evaluated by MTT assay. Several compounds exhibited potential cytotoxic activities with IC50 values below 0.10 μM. Five compounds (22g, 22h, 22l, 22s and 23a) were selected for further enzymatic determination, resulting in the discovery of 22l against ALK and ALKL1196M with IC50 values of 2.1 nM and 3.8 nM. Particularly, western blot and cell apoptosis assays identified 22l as a promising ALK inhibitor, which was capable of obviously inhibiting cellular ALK activity and inducing cell apoptosis. Eventually, molecular docking modes of 22l with ALK confirmed structural basis in accordance with the SARs analysis.

Primary lung cancer is extremely rare in children. It often presents with metastatic disease and carries a poor prognosis. Adenocarcinoma is the most common type of bronchogenic carcinoma in children and adults. Our aim was to systematically review the presenting features, approach to diagnosis and management, as well as the outcomes of primary pediatric adenocarcinoma of the lung. This systematic review was prospectively registered with PROSPERO. The following databases were searched: Medline, Embase, Web of Science, and Scopus for English language cases of primary pediatric adenocarcinoma of the lung. Forty-eight studies were included, comprising 62 patients with adenocarcinoma and 21 cases of adenocarcinoma in situ. Presenting features were nonspecific, with cough and dyspnea the main symptoms at diagnosis. The majority of patients with adenocarcinoma had metastatic disease at diagnosis. Surgery was the most common form of management. More than half the patients with adenocarcinoma had died at final follow-up, whereas 5 of 21 with adenocarcinoma in situ died. Medical management did not improve outcomes, except for two ALK receptor tyrosine kinase (ALK)-rearranged adenocarcinomas that responded to ALK inhibitor therapy alone. Primary pediatric adenocarcinoma of the lung is a rare entity which often presents with metastatic disease and portends a poor prognosis. Surgery is associated with disease-free status, although new agents such as ALK-inhibitors are able to prolong life without surgical management.

BACKGROUND: Brigatinib, a next-generation anaplastic lymphoma kinase (ALK) inhibitor, has robust efficacy in patients with ALK-positive non-small-cell lung cancer (NSCLC) that is refractory to crizotinib. The efficacy of brigatinib, as compared with crizotinib, in patients with advanced ALK-positive NSCLC who have not previously received an ALK inhibitor is unclear. METHODS: In an open-label, phase 3 trial, we randomly assigned, in a 1:1 ratio, patients with advanced ALK-positive NSCLC who had not previously received ALK inhibitors to receive brigatinib at a dose of 180 mg once daily (with a 7-day lead-in period at 90 mg) or crizotinib at a dose of 250 mg twice daily. The primary end point was progression-free survival as assessed by blinded independent central review. Secondary end points included the objective response rate and intracranial response. The first interim analysis was planned when approximately 50% of 198 expected events of disease progression or death had occurred. RESULTS: A total of 275 patients underwent randomization; 137 were assigned to brigatinib and 138 to crizotinib. At the first interim analysis (99 events), the median follow-up was 11.0 months in the brigatinib group and 9.3 months in the crizotinib group. The rate of progression-free survival was higher with brigatinib than with crizotinib (estimated 12-month progression-free survival, 67% [95% confidence interval {CI}, 56 to 75] vs. 43% [95% CI, 32 to 53]; hazard ratio for disease progression or death, 0.49 [95% CI, 0.33 to 0.74]; P

Background: The anaplastic lymphoma kinase (ALK) treatment landscape is crowded following recent ALK inhibitor approvals, and updated information on real-world treatment patterns in advanced non-small-cell lung cancer (aNSCLC) with ALK rearrangement (ALK+) is needed. Methods: This retrospective US cohort study used Flatiron Health’s longitudinal electronic health record (EHR)-derived database. Patients (≥ 18 years old) diagnosed with stage IIIB/IV aNSCLC, with documented ALK rearrangement and ≥2 visits after January 1, 2011 were followed until February 28, 2016. Patients enrolled on a clinical trial or exposed to ALK inhibitors other than crizotinib or ceritinib were excluded. Treatment patterns, time and type of biomarker testing, and overall survival (OS) were analyzed. Results: Median age (n = 300) was 62.5 years; 55% female; 48% non-smokers; 8.7% central nervous system (CNS) metastases at diagnosis. Overall, 73% and 86% received their first ALK biomarker test before/at diagnosis, or before/during first-line treatment, respectively. In total, 90.0%, 78.1%, and 74.7% received first-, second-, and third-line therapy, respectively. Most patients received ALK-targeted treatment; 62% received crizotinib, of which 21% reported a dose reduction. Progression was the most common reason for crizotinib (78%) and ceritinib (41%) discontinuation. Median OS was 29.4 months (95% CI =24.7–39.6) overall; 27.1 months (95% CI =22.0–35.0) in patients with CNS metastases, and 36.9 months (95% CI =25.1–not reached) without. Conclusions: Despite widespread crizotinib use in patients with ALK+ aNSCLC, a high proportion of patients progressed. Ongoing analyses of EHR-derived cohorts are valuable in assessing real-world testing rates and therapeutic use of ALK inhibitors.

ALK-rearranged lung cancer defines a distinctive molecular cohort of patients whose outcomes are significantly improved by the availability of ALK inhibitors. Thus, it is imperative for clinicians to screen appropriate patients for this driver mutation with a molecular testing platform capable of capturing all ALK fusions. Here, we report a novel VKORC1L1-ALK fusion and an ALK T1151K resistance mutation detected in a lung cancer patient who had been on crizotinib for over 8 years. Alectinib induced a dramatic response in this patient demonstrating its clinical activity against T1151K. This case illustrates the importance of performing repeat biopsy to explore mechanism(s) of resistance when patients experience disease progression on an ALK inhibitor. The approach has a direct therapeutic impact particularly when an ALK resistance mutation is identified.

The National Institute for Health and Care Excellence (NICE) invited the company that manufactures ceritinib (Zykadia®, Novartis) to submit evidence on the clinical and cost effectiveness of the drug, as a first-line treatment for adults with anaplastic lymphoma kinase (ALK)-positive (+) advanced non-small-cell lung cancer (NSCLC), as part of the Institute’s single technology appraisal (STA) process. The CRD (Centre for Reviews and Dissemination) and CHE (Centre for Health Economics) Technology Assessment Group at the University of York was commissioned to act as the Evidence Review Group (ERG). This paper describes the Company’s submission (CS), the ERG review and NICE’s subsequent decisions. The evidence submitted in support of ceritinib, as the first-line treatment in ALK+ advanced NSCLC, was a phase III, international, multicentre, open-label randomised controlled trial (RCT) comparing ceritinib with pemetrexed/cisplatin plus pemetrexed maintenance therapy (chemotherapy [CT] group). The results indicated that ceritinib prolonged progression-free survival (PFS) compared with CT. The only comparator considered in the CS was crizotinib. The evidence selected in support of crizotinib was PROFILE 1014, an open-label RCT of crizotinib, compared with pemetrexed/cisplatin CT (without maintenance therapy), in previously untreated advanced or metastatic ALK+ NSCLC. The design and population of PROFILE 1014 was similar to that of ASCEND-4, though there were some differences between the trials. The Company considered it not possible to perform an ‘anchor-based’ analysis of first-line ceritinib and crizotinib, and presented a Matching-Adjusted Indirect Comparison (MAIC) of ceritinib and crizotinib using only the ALK inhibitor arm of ASCEND-4 and PROFILE 1014. The indirect comparison suggests that ceritinib may be more effective in prolonging PFS than crizotinib. The ERG agreed that an indirect comparison using only the ALK inhibitor arm of the trials was the only option available in the present assessment; however, a number of limitations and potential bias were identified in this analysis. The Company’s model estimated that ceritinib was cost effective when compared with crizotinib. However, the ERG highlighted several concerns with the Company’s analysis; the ERG’s preferred base case estimated an incremental cost-effectiveness ratio of £69,255 per quality-adjusted life-year (no patient access scheme [PAS] included). The ERG considered the economic analysis to be sensitive to changes in assumption used, partly due to the due to the immaturity of the overall survival data from trials, which leads to uncertainty around the extrapolation used. The NICE Appraisal Committee concluded that ceritinib is recommended, within its marketing authorisation, as an option for untreated ALK+ advanced NSCLC in adults, if the Company provides it with the discount agreed in the PAS.

The use of tyrosine kinase inhibitors (TKI) with activity against ALK, ROS1, or TRKA–C can result in significant clinical benefit in patients with diverse tumors harboring ALK, ROS1, or NTRK1–3 rearrangements; however, resistance invariably develops. The emergence of on-target kinase domain mutations represents a major mechanism of acquired resistance. Solvent-front substitutions such as ALKG1202R, ROS1G2032R or ROS1D2033N, TRKAG595R, and TRKCG623R are among the most recalcitrant of these mechanisms. Repotrectinib (TPX-0005) is a rationally designed, low-molecular-weight, macrocyclic TKI that is selective and highly potent against ROS1, TRKA–C, and ALK. Importantly, repotrectinib exhibits activity against a variety of solvent-front substitutions in vitro and in vivo. As clinical proof of concept, in an ongoing first-in-human phase I/II trial, repotrectinib achieved confirmed responses in patients with ROS1 or NTRK3 fusion–positive cancers who had relapsed on earlier-generation TKIs due to ROS1 or TRKC solvent-front substitution-mediated resistance. Significance: Repotrectinib (TPX-0005), a next-generation ROS1, pan-TRK, and ALK TKI, overcomes resistance due to acquired solvent-front mutations involving ROS1, NTRK1–3, and ALK. Repotrectinib may represent an effective therapeutic option for patients with ROS1-, NTRK1–3-, or ALK-rearranged malignancies who have progressed on earlier-generation TKIs. Cancer Discov; 8(10); 1227–36. ©2018 AACR. This article is highlighted in the In This Issue feature, p. 1195

Entrectinib (ENC) is a potent orally available anaplastic lymphoma kinase (ALK) inhibitor. In 10 July 2017, biotechnology company (Ignyta) announced that granted orphan drug designation approval was given by the FDA to ENC for “treatment of NTRK fusion-positive solid tumors”. A validated LC-MS/MS methodology was developed for ENC quantification in human plasma matrix. The supposed method characterized by high speed, specificity and sensitivity. This established method was applied for metabolic degradation assessment of ENC. Reversed stationary phase (C18 column) and elution mobile phase (48% 10 mM ammonium formate in H2O (pH: 4.2 adjusted by adding few drops of formic acid): 52% ACN) were utilized for chromatographic resolution of ENC and lapatinib as internal standard (IS). Total elution time, flow rate and injection volume were 4 min., 0.25 mL/min., and 5 μL, respectively. Electrospray ionization source was used for ions generation, while positive multiple reactions monitoring (MRM) mode was used for ion analysis. The data of calibration curve of ENC in human plasma was linear in the range of 5–500 ng/mL with correlation coefficient (r2) >0.999. LOQ and LOD for ENC were 2.17 ng/mL and 0.71 ng/mL, respectively. Inter-day and intra-day precision and accuracy were 97.52 to 101.83%, and 0.38 to 1.32%, respectively. Intrinsic clearance (Clint) and in vitro half-life (t1/2) were equal to 15.67 mL/min/kg and 9.1 min, respectively. To our knowledge, this is considered the first method for ENC quantification in human plasma and its metabolic degradation assessment.

The targeted treatment of advanced non-small cell lung cancer (NSCLC) harboring genomic rearrangement of ALK is a paradigm for personalized oncology. More than 15 different ALK fusion partners have been discovered in NSCLC patients. Most of these ALK fusions responded well to the ALK inhibitor crizotinib. Crizotinib is an oral MET/ALK inhibitor used as first-line therapy in the treatment of advanced NSCLC harboring ALK rearrangement. An understanding of the mechanisms by which tumors harbor primary drug resistance or acquired resistance to targeted therapies is critical for predicting which patients will respond to a specific therapy and for the identification of additional targetable pathways to maximize clinical benefits. Cap methyltransferase 1(CMTR1) also known as hMTr1, which is translate a human cap1 2ʹ-o-ribose methyltransferase. Here, we report the newly found ALK fusion, CMTR1-ALK, in a patient who has no response to the ALK inhibitor crizotinib. The results remind us that detecting ALK status is important, but that determining the ALK fusion type and function may be more important for patient.

Advances in chromosomally rearranged ALK positive non-small cell lung cancer have been dramatic in only the last few years. Survival times have improved dramatically due to the introduction of ever more efficacious ALK inhibitors. These improvements have been due largely to improvements in blood-brain barrier penetration and the breadth of ligand binding pocket mutations against which the drugs are effective. However, the advances maybe slow due to the frequency of cancers with compound resistance mutations are appearing, suggesting the need to develop multiple ALK inhibitors to target different compound mutations.Another research area that promises to provide further gains is the use of drug combinations, with an ALK inhibitor combined with a drug targeting a “second driver” to overcome resistance. In this review, the range of secondary targets for ALK+ lung cancer and the potential for their clinical success are reviewed.

For non-small cell lung cancer (NSCLC) patients with ALK-rearranged tumors, treatment with ALK inhibitors can improve outcomes. However, clinical resistance typically develops over time, and in the majority of cases resistance mechanisms are ALK-independent. We generated tumor cell cultures from multiple regions of an ALK-rearranged clinical tumor specimen, and deployed functional drug screens to identify modulators of resistance to ALK inhibitors. This identified a role for PI3Kβ and EGFR in regulating resistance to ALK inhibition. Furthermore, inhibition of ALK elicited activation of EGFR, and inhibition of PI3Kβ rescued EGFR-mediated ALK inhibitor resistance. In ALK-rearranged primary cultures, cell lines and in vivo xenograft models, combined inhibition of ALK and PI3Kβ prevented compensatory MAPK and PI3K-AKT pathway reactivation and selectively targeted the cancer cells. The combinatorial effect was seen even in the background of TP53 mutations and in epithelial-mesenchymal transformed cells. In conclusion, combinatorial ALK and PI3Kβ inhibitor treatment carries promise as a treatment for ALK-rearranged NSCLC.

Alcohol use disorder (AUD) has a complex pathogenesis, making it a difficult disorder to treat. Identifying relevant signaling pathways in the brain may be useful for finding new pharmacological targets to treat AUD. The receptor tyrosine kinase anaplastic lymphoma kinase (ALK) activates the transcription factor STAT3 in response to ethanol in cell lines. Here, we show ALK activation and upregulation of known STAT3 target genes (Socs3, Gfap and Tnfrsf1a) in the prefrontal cortex (PFC) and ventral hippocampus (HPC) of mice after 4 days of binge-like ethanol drinking. Mice treated with the STAT3 inhibitor stattic drank less ethanol than vehicle-treated mice, demonstrating the behavioral importance of STAT3. To identify novel ethanol-induced target genes downstream of the ALK and STAT3 pathway, we analyzed the NIH LINCS L1000 database for gene signature overlap between ALK inhibitor (alectinib and NVP-TAE684) and STAT3 inhibitor (niclosamide) treatments on cell lines. These genes were then compared with differentially expressed genes in the PFC of mice after binge-like drinking. We found 95 unique gene candidates, out of which 57 had STAT3 binding motifs in their promoters. We further showed by qPCR that expression of the putative STAT3 genes Nr1h2, Smarcc1, Smarca4 and Gpnmb were increased in either the PFC or HPC after binge-like drinking. Together, these results indicate activation of the ALK-STAT3 signaling pathway in the brain after binge-like ethanol consumption, identify putative novel ethanol-responsive STAT3 target genes, and suggest that STAT3 inhibition may be a potential method to reduce binge drinking in humans.

Since 2011, with the approval of crizotinib and subsequent approval of four additional targeted therapies, anaplastic lymphoma kinase (ALK) inhibitors have become important treatments for a subset of patients with lung cancer. Each generation of ALK inhibitor showed improvements in terms of central nervous system (CNS) penetration and potency against wild-type (WT) ALK, yet a key continued limitation is their susceptibility to resistance from ALK active-site mutations. The solvent front mutation (G1202R) and gatekeeper mutation (L1196M) are major resistance mechanisms to the first two generations of inhibitors while patients treated with the third-generation ALK inhibitor lorlatinib often experience progressive disease with multiple mutations on the same allele (mutations in cis, compound mutations). TPX-0131 is a compact macrocyclic molecule designed to fit within the ATP-binding boundary to inhibit ALK fusion proteins. In cellular assays, TPX-0131 was more potent than all five approved ALK inhibitors against WT ALK and many types of ALK resistance mutations, e.g., G1202R, L1196M, and compound mutations. In biochemical assays, TPX-0131 potently inhibited (IC50

Background Lorlatinib is a third-generation anaplastic lymphoma kinase (ALK)/c-ros oncogene 1 (ROS1) tyrosine kinase inhibitor (TKI) with efficacy in patients with ALK-rearranged non-small-cell lung cancer (NSCLC) previously treated with a second-generation ALK inhibitor or with first- and second-generation ALK inhibitors. We examined the cost-effectiveness of second- or third-line+ (2L+ or 3L+) lorlatinib in Sweden, versus chemotherapy. Methods A partitioned survival model with three health states (progression free, progressed, or death) was used. Lorlatinib relative efficacy versus chemotherapy was derived using unanchored matching adjusted indirect treatment comparisons from a phase 2 clinical trial. Utility data were derived from the same trial and published studies. Costs (year 2019) were obtained from Swedish national data. Costs and benefits were discounted at 3% per annum using a societal perspective (base case). Model robustness was evaluated with deterministic and probabilistic sensitivity analyses. Results For 2L+, the average discounted total quality-adjusted life year (QALY) gain was 1.29 years. Total incremental costs were Swedish krona (SEK) 731,791, resulting in an incremental cost-effectiveness ratio (ICER) of SEK 566,278 per QALY gained. Non-discounted survival gain amounted to 1.94 years. For 3L+, the average discounted total QALY gain was 1.25 years. Total incremental costs were SEK 754,801, resulting in an ICER of SEK 603,934 per QALY gained. Non-discounted survival gain was 1.88 years. Sensitivity analyses were consistent. Conclusions ICERs ranged from SEK 421,000 to SEK 384,066 less than the boundary for a cost-effective treatment for a high-severity disease in Sweden (SEK 988,000), suggesting 2L+ or 3L+ lorlatinib is a cost-effective treatment for ALK-positive NSCLC versus chemotherapy. Supplementary Information The online version contains supplementary material available at 10.1007/s40273-021-01015-8.

The ALK inhibitors are promising therapeutic agents against lung cancer harboring ALK fusion genes and are currently under development up to the third generation. However, its therapeutic effects are reported to be affected by differences in ALK variants and co-occurrent mutations. Materials and Methods; We experienced an autopsy case of an ALK-positive lung cancer patient who showed primary resistance to three generations of ALK inhibitors. The poor survival time of the case was 14 months. To reveal the mechanism of primary resistance to three generations of ALK inhibitors, we performed next generation sequencing for 12 specimes obtained from an autopsy with covering whole exons of 53 significantly mutated, lung cancer-associated genes and amplicon-based target RNA sequenceing for the ALK fusion gene. The NGS analysis revealed a rare variant.3 of ALK fusion, in which 30 bp of base was inserted at the end of ALK intron.19 and was associated with EML exon.6 [E6_ins30A20] and a co-occurrent oncogenic PIK3CA E542K mutation in all specimens. Structural analysis of the fusion protein ALK [E6_ins30A20] showed no interferance with the binding of ALK inhibitors to the kinase domain. The NGS analysis of primary and metastatic lesions obtained from an autopsy revealed a co-occurrent oncogenic PIK3CA E542K mutation in all specimens. The constitutive activation of PI3K-Akt signal by PIK3CA E542K mutation occurred downstream of ALK signaling pathway, could lead to primary resistance to ALK inhibitors in all generations.

Neuroblastoma (NB) is the most common extracranial solid tumour in children. NB is highly heterogeneous and is comprised of a mixture of neuroblastic cancer cells and stromal cells. We previously reported that N‐type cells (neuroblastic cells) and S‐type cells (substrate‐adherent cells) in the SK‐N‐SH cell line shared almost identical genetic backgrounds. Sublines of N‐ and S‐type cells were isolated from an early passage (P35) of SK‐N‐SH. Sequencing analysis revealed that all sublines harboured the anaplastic lymphoma kinase (ALK) F1174L mutation, indicating that they were tumour derived. Surprisingly, over 74% resembled S‐type cells. In coculture experiments, S‐type cells protected N‐type cells from apoptosis induced by the oncogenic ALK inhibitor TAE684. Western blotting analyses showed that ALK, protein kinase A (AKT) and STAT3 signalling were stimulated in the cocultures. Furthermore, the conditioned medium from S‐type cells activated these downstream signalling molecules in the N‐type cells. The activation of STAT3 in the N‐type cells was ALK‐independent, while AKT was regulated by the ALK activation status. To identify the responsible soluble factors, we used a combination of transcriptomic and proteomic analysis and found that plasminogen activator inhibitor 1, secreted protein acidic and cysteine rich, periostin and galectin‐1 were potential mediators of STAT3 signalling. The addition of recombinant proteins to the tumour cells treated with the ALK inhibitor partially enhanced cell viability. Overall, the tumour‐derived S‐type cells prevented apoptosis in the N‐type cells via ALK‐independent STAT3 activation triggered by secreted factors. The inhibition of these factors in combination with ALK inhibition could provide a new direction for targeted therapies to treat high‐risk NB., Neuroblastoma S‐type cells promote survival of N‐type tumor cells through cell‐to‐cell contact, which is associated with upregulation of p‐STAT3 in N‐type cells. Independently, S‐type cells secrete trophic factors that promote the survival of N‐type cells. Trophic factors could act either by stimulating p‐STAT3, or by upregulating p‐AKT and p‐ERK1/2 through an p‐ALK‐dependent pathway. The inhibition of these factors in combination with ALK inhibition could provide a new strategy for treatment of high‐risk neuroblastoma.

Metastatic pheochromocytoma and paraganglioma (PPGL) are rare diseases with dismal prognosis and standard therapies are lacking. We herein report the first case of a germline anaplastic lymphoma kinase (ALK) mutation in a patient with chemorefractory metastatic pheochromocytoma in the absence of mutations of known PPGL-associated predisposing genes. Therapy with the ALK inhibitor (ALKi) brigatinib led to dramatic and durable disease remission, despite previous disease progression on the ALKi alectinib. This case underscores the potential clinical use of molecular profiling in rare diseases with limited treatment options and suggests that the ALK-R1192P point mutation might predict sensitivity to brigatinib., Highlights • First case of a germline ALK mutation in a patient with metastatic pheochromocytoma. • Durable response to the ALKi brigatinib after disease progression on combination chemotherapy and alectinib. • A germline ALK mutation in a patient with pheochromocytoma in the absence of mutations of known PPGL-associated genes.

Background More than 80% of anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma (ALCL) patients harbor the (nucleophosmin) NPM1-ALK fusion gene t(2;5) chromosomal translocation. We evaluated the preclinical and clinical efficacy of ceritinib treatment of this aggressive lymphoma. Materials and methods We studied the effects of ceritinib treatment in NPM1-ALK+ T-cell lymphoma cell lines in vitro and on tumor size and survival advantage in vivo utilizing tumor xenografts. We treated an NPM1-ALK+ ALCL patient with ceritinib. We reviewed all hematologic malignancies profiled by a large hybrid-capture next-generation sequencing (NGS)-based comprehensive genomic profiling assay for ALK alterations. Results In our in vitro experiments, ceritinib inhibited constitutive activation of the fusion kinase NPM1-ALK and downstream effector molecules STAT3, AKT, and ERK1/2, and induced apoptosis of these lymphoma cell lines. Cell cycle analysis following ceritinib treatment showed G0/G1 arrest with a concomitant decrease in the percentage of cells in S and G2/M phases. Further, treatment with ceritinib in the NPM1-ALK+ ALCL xenograft model resulted in tumor regression and improved survival. Of 19 272 patients with hematopoietic diseases sequenced, 58 patients (0.30%) harbored ALK fusions that include histiocytic disorders, multiple myeloma, B-cell neoplasms, Castleman's disease, and juvenile xanthogranuloma. A multiple relapsed NPM1-ALK+ ALCL patient treated with ceritinib achieved complete remission with ongoing clinical benefit to date, 5 years after initiation of therapy. Conclusions This ceritinib translational study in NPM1-ALK+ ALCL provides a strong rationale for a prospective study of ceritinib in ALK+ T-cell lymphomas and other ALK+ hematologic malignancies., Graphical abstract, Highlights • The fusion kinase NPM1-ALK is constitutively activated in a ligand-independent manner. • Preclinically, ceritinib inhibits fusion kinase activity, along with inducing apoptosis in NPM1-ALK+ ALCL cells. • Tumors beyond ALK+ lymphomas such as histiocytic disorders, multiple myeloma, B-cell neoplasms, harbor ALK fusions. • Ceritinib therapy documented durable complete response in an NPM1-ALK+ ALCL patient corroborating our preclinical results.

Brigatinib; NSCLC; Inhibidor de la tirosina quinasa Brigatinib; NSCLC; Inhibidor de la tirosina quinasa Brigatinib; NSCLC; Tyrosine kinase inhibitor Background The next-generation ALK inhibitor brigatinib is approved for use in patients with ALK inhibitor-naïve ALK-positive advanced NSCLC and in patients previously treated with crizotinib. A phase II trial showed that brigatinib is active in patients with ALK-positive metastatic NSCLC (mNSCLC) who had progressed on prior crizotinib (response rate 56 %, median PFS 16.7 months, median OS 34.1 months). We report final data from the UVEA-Brig study of brigatinib in ALK inhibitor-pretreated ALK-positive mNSCLC in clinical practice. Methods UVEA-Brig was a retrospective chart review of patients treated with brigatinib in Italy, Norway, Spain and the UK in an expanded access program. Adults with ALK-positive mNSCLC, including those with brain lesions, resistant to or intolerant of ≥1 prior ALK inhibitor and ECOG performance status ≤3 were eligible. Patients received brigatinib 180 mg once daily with a 7-day lead-in at 90 mg. The objectives were to describe patient characteristics, clinical disease presentation, treatment regimens used and clinical outcomes. Results Data for 104 patients (male: 43 %; median age: 53 [29–80] years; ECOG performance status 0/1/2/3: 41/41/10/5 %; brain/CNS metastases: 63 %) were analyzed. Patients had received a median of 2 (1–6) lines of systemic therapy prior to brigatinib (37.5 % received ≥3) and a median of 1 (1–5) lines of prior ALK inhibitor-containing therapy (crizotinib 83.6 %; ceritinib 50.0 %; alectinib 6.7 %; lorlatinib 4.8 %). At the time of analysis, 77 patients had discontinued brigatinib. Overall, the response rate was 39.8 %, median PFS was 11.3 (95 % CI:8.6–12.9) months and median OS was 23.3 (95 % CI: 16.0–NR) months. Four patients discontinued brigatinib treatment due to adverse events. 53 patients received systemic therapy after brigatinib, 42 with an ALK inhibitor (lorlatinib, n = 34). Conclusions These real-world data indicate the activity and tolerability of brigatinib in patients with ALK-positive mNSCLC who were more heavily pretreated than patients included in clinical trials. This study was funded by Takeda Pharmaceuticals International AG, Zurich, Switzerland.

Patients diagnosed with anaplastic large cell lymphoma (ALCL) are still treated with toxic multi-agent chemotherapy and as many as 25-50% of patients relapse. To understand disease pathology and to uncover novel targets for therapy, we performed whole-exome sequencing of anaplastic lymphoma kinase (ALK)+ ALCL, as well as gene-set enrichment analysis. This revealed that the T-cell receptor and Notch pathways were the most enriched in mutations. In particular, variant T349P of NOTCH1, which confers a growth advantage to cells in which it is expressed, was detected in 12% of ALK+ and ALK– ALCL patients’ samples. Furthermore, we demonstrated that NPM-ALK promotes NOTCH1 expression through binding of STAT3 upstream of NOTCH1. Moreover, inhibition of NOTCH1 with γ- secretase inhibitors or silencing by short hairpin RNA leads to apoptosis; cotreatment in vitro with the ALK inhibitor crizotinib led to additive/synergistic antitumor activity suggesting that this may be an appropriate combination therapy for future use in the circumvention of ALK inhibitor resistance. Indeed, crizotinib-resistant and -sensitive ALCL were equally sensitive to γ-secretase inhibitors. In conclusion, we show a variant in the extracellular domain of NOTCH1 that provides a growth advantage to cells and confirm the suitability of the Notch pathway as a second-line druggable target in ALK+ ALCL.

Treatment of advanced stage anaplastic lymphoma kinase (ALK) positive non-small cell lung cancer (NSCLC) with ALK tyrosine kinase inhibitors (TKIs) has been shown to be superior to standard platinum-based chemotherapy. However, secondary progress of disease frequently occurs under ALK inhibitor treatment. The clinical impact of re-biopsies for treatment decisions beyond secondary progress is, however, still under debate. Here, we report on two novel subsequent polyclonal on-and off-target resistance mutations in a patient with ALK-fused NSCLC under ALK inhibitor treatment. A 63-year-old male patient with an advanced stage EML4-ALK fused pulmonary adenocarcinoma was initially successfully treated with the second-generation ALK inhibitor alectinib and upon progressions subsequently with brigatinib, lorlatinib and chemoimmunotherapy (CIT). Progress to alectinib was associated with a so far undescribed ALK mutation (p.A1200_G1201delinsW) which was, however, tractable by brigatinib. An off-target KRAS-mutation (p.Q61K) occurred in association with subsequent progression under second-line TKI treatment. Third-line lorlatinib showed limited efficacy but chemoimmunotherapy resulted in disappearance of the KRAS mutant clone and clinical tumor control for another eight months. In conclusion, we suggest molecular profiling of progressive tumor disease also for ALK-positive NSCLC to personalize treatment in a subgroup of ALK-positive patients. CA extern

Purpose: In the literature there are conflicting data about the treatment efficacy of anaplastic lymphoma kinase (ALK) translocation positive non-small cell lung cancer (NSCLC) patients according to ALK fusion variants. We aimed to study the impact of ALK fusion variants on the survival of first-line crizotinib-treated NSCLC patients. Methods: 101 locally advanced or metastatic ALK positive NSCLC patients treated with first-line crizotinib between January 2013 and December 2019 were retrospectively evaluated. We studied ALK fusion variants in 38 of those patients with adequate tumor tissue with reverse transcription polymerase chain reaction (RT PCR). Patients having ALK fusion variant 1 (v1) and non-variant 1 (non-v1) were compared for survival and response to crizotinib. Results: Median age was 52.5 years (range 35-74), and 22 of 38 patients were male (57.9%). EML-4 ALK v1 was seen in 26 patients (68.4%) and 12 were non-v1 (variant 3a/b in 6, and non-EML-4 ALK variants in 6 patients). Objective response rate was 60.5% in all patients, whereas it was 61.5% in v1 and 58.3% in non-v1 group. Median progression-free survival (PFS) and overall survival (OS) were similar. Median PFS was 13.1 months in v1, and 12.4 months in non-v1 (p=0.232). Median OS was 23.2 months in v1, and 19.4 months non-v1 (p=0.493). Conclusion: ALK v1 and non-v1 patients had the same OS and PFS after first-line crizotinib treatment, however there was a trend for v1 group for better OS. Turkish Oncology Group

Approximately 5–7% of non–small cell lung cancer (NSCLC) cases harbor an anaplastic lymphoma kinase (ALK) fusion gene and may benefit from ALK inhibitor therapy. To detect ALK fusion genes, we developed a novel test using reverse transcription polymerase chain reaction (RT-PCR) for the ALK kinase domain (KD). Since ALK expression is mostly silenced in the adult with the exception of neuronal tissue, the normal lung tissue, mesothelial lining, and inflammatory cells are devoid of ALK transcript, making ALK KD RT-PCR an ideal surrogate test for ALK fusion transcripts in lung or pleural effusion. The test was designed with a short PCR product (197 bp) to work for both malignant pleural effusion (MPE) and formalin-fixed, paraffin-embedded (FFPE) NSCLC samples. Using ALK IHC as a reference, the sensitivity of the test was 100% for both MPE and FFPE. The specificity was 97.6% for MPE and 97.4% for FFPE. Two false positive cases were found. One was a metastatic brain lesion which should be avoided in the future due to intrinsic ALK expression in the neuronal tissue. The other one resulted from ALK gene amplification. Due to potential false positivity, subsequent confirmation tests such as fluorescence in situ hybridization or multiplex PCR would be preferable. Nevertheless, the test is simple and inexpensive with no false negativity, making it a desirable screening test. It also offers an advantage over multiplex RT-PCR with the capability to detect novel ALK fusions. Indeed through the screening test, we found a novel ALK fusion partner (sperm antigen with calponin homology and coiled-coil domains 1 like gene, SPECC1L) with increased sensitivity to crizotinib in vitro. In summary, a novel RNA-based ALK KD analysis was developed for ALK rearrangement screening in MPE and FFPE specimens of NSCLC. This simple inexpensive test can be implemented as routine diagnostics.

Around 3-7% of patients with non-small cell lung cancer (NSCLC), which represent 85% of diagnosed lung cancers, have a rearrangement in the ALK gene that produces an abnormal activity of the ALK protein cell signaling pathway. The developed ALK tyrosine kinase inhibitors (TKIs), such as crizotinib, ceritinib, alectinib, brigatinib and lorlatinb present good performance treating ALK+ NSCLC, although all patients invariably develop resistance due to ALK secondary mutations or bypass mechanisms. In the present study, we compare the potential differences between brigatinib and alectinib's mechanisms of action as first-line treatment for ALK+ NSCLC in a systems biology-based in silico setting. Therapeutic performance mapping system (TPMS) technology was used to characterize the mechanisms of action of brigatinib and alectinib and the impact of potential resistances and drug interferences with concomitant treatments. The analyses indicate that brigatinib and alectinib affect cell growth, apoptosis and immune evasion through ALK inhibition. However, brigatinib seems to achieve a more diverse downstream effect due to a broader cancer-related kinase target spectrum. Brigatinib also shows a robust effect over invasiveness and central nervous system metastasis-related mechanisms, whereas alectinib seems to have a greater impact on the immune evasion mechanism. Based on this in silico head to head study, we conclude that brigatinib shows a predicted efficacy similar to alectinib and could be a good candidate in a first-line setting against ALK+ NSCLC. Future investigation involving clinical studies will be needed to confirm these findings. These in silico systems biology-based models could be applied for exploring other unanswered questions.

Aims: Premarketing clinical trials are typically conducted under controlled conditions and in selected study populations, so real-world information on the utilization of new cancer drugs is limited. We aimed to explore the potential of German claims data in this regard, exemplified by the ALK inhibitor crizotinib, used in non-small-cell lung cancer therapy. Materials & methods: We identified patients treated with crizotinib in the German Pharmacoepidemiological Research Database (2004–2017; 20% of the German population) and assessed patient characteristics, treatment and survival. Results: We identified 348 crizotinib-treated patients (56% female; 25% first-line users). After 2 years, overall survival was 48%, with higher survival in men than in women (58 vs 40%). Overall, 76% of patients discontinued crizotinib treatment. Of those, 41% received another ALK inhibitor afterward. Conclusion: The results underline the potential of German claims data for real-world monitoring of oncological drug utilization.

Alterations in the ALK gene are found in 8% of familial and sporadic neuroblastoma cases. The frequency of ALK aberrations in high-risk neuroblastomas is 14% (10% point mutation and 4% amplification) at the time of diagnosis, with increasing number of aberrations at tumor relapse (Bresler et al., 2011). Despite recent advancements in ALK-targeted therapies, ALK inhibitor potency eventually diminishes, and drug resistance emerges after initial successful treatment. To improve drug efficacy and overcome resistance to ALK inhibitors, combined regimens with chemotherapy or other targeted therapies showed superior anti-tumor effects in preclinical and early clinical studies, making it a promising strategy for neuroblastoma treatment.

Crizotinib was a first generation of ALK tyrosine kinase inhibitor approved for the treatment of ALK-positive non-small-cell lung carcinoma (NSCLC) patients. COMPARE and cluster analyses of transcriptomic data of the NCI cell line panel indicated that genes with different cellular functions regulated the sensitivity or resistance of cancer cells to crizotinib. Transcription factor binding motif analyses in gene promoters divulged two transcription factors possibly regulating the expression of these genes, i.e., RXRA and GATA1, which are important for leukemia and erythroid development, respectively. COMPARE analyses also implied that cell lines of various cancer types displayed varying degrees of sensitivity to crizotinib. Unexpectedly, leukemia but not lung cancer cells were the most sensitive cells among the different types of NCI cancer cell lines. Re-examining this result in another panel of cell lines indeed revealed that crizotinib exhibited potent cytotoxicity towards acute myeloid leukemia and multiple myeloma cells. P-glycoprotein-overexpressing CEM/ADR5000 leukemia cells were cross-resistant to crizotinib. NCI-H929 multiple myeloma cells were the most sensitive cells. Hence, we evaluated the mode of action of crizotinib on these cells. Although crizotinib is a TKI, it showed highest correlation rates with DNA topoisomerase II inhibitors and tubulin inhibitors. The altered gene expression profiles after crizotinib treatment predicted several networks, where TOP2A and genes related to cell cycle were downregulated. Cell cycle analyses showed that cells incubated with crizotinib for 24 h accumulated in the G2M phase. Crizotinib also increased the number of p-H3(Ser10)-positive NCI-H929 cells illustrating crizotinib’s ability to prevent mitotic exit. However, cells accumulated in the sub-G0G1 fraction with longer incubation periods, indicating apoptosis induction. Additionally, crizotinib disassembled the tubulin network of U2OS cells expressing an α-tubulin-GFP fusion protein, preventing migration of cancer cells. This result was verified by in vitro tubulin polymerization assays. In silico molecular docking also revealed a strong binding affinity of crizotinib to the colchicine and Vinca alkaloid binding sites. Taken together, these results demonstrate that crizotinib destabilized microtubules. Additionally, the decatenation assay showed that crizotinib partwise inhibited the catalytic activity of DNA topoisomerase II. In conclusion, crizotinib exerted kinase-independent cytotoxic effects through the dual inhibition of tubulin polymerization and topoisomerase II and might be used to treat not only NSCLC but also multiple myeloma.

Lung cancer is one of the most common malignant neoplasms. As a result of the disease’s progression, patients may develop metastases to the central nervous system. The prognosis in this location is unfavorable; untreated metastatic lesions may lead to death within one to two months. Existing therapies—neurosurgery and radiation therapy—do not improve the prognosis for every patient. The discovery of Epidermal Growth Factor Receptor (EGFR)—activating mutations and Anaplastic Lymphoma Kinase (ALK) rearrangements in patients with non-small cell lung adenocarcinoma has allowed for the introduction of small-molecule tyrosine kinase inhibitors to the treatment of advanced-stage patients. The Epidermal Growth Factor Receptor (EGFR) is a transmembrane protein with tyrosine kinase-dependent activity. EGFR is present in membranes of all epithelial cells. In physiological conditions, it plays an important role in the process of cell growth and proliferation. Binding the ligand to the EGFR causes its dimerization and the activation of the intracellular signaling cascade. Signal transduction involves the activation of MAPK, AKT, and JNK, resulting in DNA synthesis and cell proliferation. In cancer cells, binding the ligand to the EGFR also leads to its dimerization and transduction of the signal to the cell interior. It has been demonstrated that activating mutations in the gene for EGFR-exon19 (deletion), L858R point mutation in exon 21, and mutation in exon 20 results in cancer cell proliferation. Continuous stimulation of the receptor inhibits apoptosis, stimulates invasion, intensifies angiogenesis, and facilitates the formation of distant metastases. As a consequence, the cancer progresses. These activating gene mutations for the EGFR are present in 10–20% of lung adenocarcinomas. Approximately 3–7% of patients with lung adenocarcinoma have the echinoderm microtubule-associated protein-like 4 (EML4)/ALK fusion gene. The fusion of the two genes EML4 and ALK results in a fusion gene that activates the intracellular signaling pathway, stimulates the proliferation of tumor cells, and inhibits apoptosis. A new group of drugs—small-molecule tyrosine kinase inhibitors—has been developed; the first generation includes gefitinib and erlotinib and the ALK inhibitor crizotinib. These drugs reversibly block the EGFR by stopping the signal transmission to the cell. The second-generation tyrosine kinase inhibitor (TKI) afatinib or ALK inhibitor alectinib block the receptor irreversibly. Clinical trials with TKI in patients with non-small cell lung adenocarcinoma with central nervous system (CNS) metastases have shown prolonged, progression-free survival, a high percentage of objective responses, and improved quality of life. Resistance to treatment with this group of drugs emerging during TKI therapy is the basis for the detection of resistance mutations. The T790M mutation, present in exon 20 of the EGFR gene, is detected in patients treated with first- and second-generation TKI and is overcome by Osimertinib, a third-generation TKI. The I117N resistance mutation in patients with the ALK mutation treated with alectinib is overcome by ceritinib. In this way, sequential therapy ensures the continuity of treatment. In patients with CNS metastases, attempts are made to simultaneously administer radiation therapy and tyrosine kinase inhibitors. Patients with lung adenocarcinoma with CNS metastases, without activating EGFR mutation and without ALK rearrangement, benefit from immunotherapy. This therapeutic option blocks the PD-1 receptor on the surface of T or B lymphocytes or PD-L1 located on cancer cells with an applicable antibody. Based on clinical trials, pembrolizumab and all antibodies are included in the treatment of non-small cell lung carcinoma with CNS metastases.

Lorlatinib is a third-generation ALK inhibitor that can overcome the largest number of acquired ALK resistance mutations, including the solvent-front mutation G1202R. Here, we report, for the first time, a novel, sequentially-evolved EML4-ALK variant 3 G1202R/S1206Y double mutation in cis detected in a patient with ALK-positive NSCLC after disease progression on sequential crizotinib, alectinib, and then lorlatinib. Three-dimensional computer modeling of this double mutation and other G1202R-based double mutations with lorlatinib (ALK G1202R/L1196M, ALK G1202R/F1174C, ALK G1202R/l1198F, ALK G1202R/G1269A) were provided to reveal how these double mutations may confer resistance to lorlatinib through diverse steric hindrances in the ALK kinase domain. In addition, we performed a comprehensive literature review on published acquired double or triple ALK mutations that are resistant to lorlatinib from both patient samples and in vitro mutagenesis experiments.

The clinical success of ALK inhibition through targeted therapies is manifestly evident in ALK-rearranged non-small cell lung cancer (NSCLC). Deciphering resistance mechanisms to targeted agents in ALK-positive NSCLC has been crucial to develop novel inhibitors and novel treatment strategies, with a continued and reciprocal exchange of evidence between clinical and preclinical research. With the advent of the third-generation ALK inhibitor lorlatinib in the clinical setting, as well as with the availability of second-generation agents (e.g., alectinib) in the upfront treatment of ALK-rearranged NSCLC, novel scenarios of resistance have recently emerged. In parallel, moving ALK inhibition in disease setting earlier than the advanced/metastatic one would likely demand new concepts and methodologies. Whereas the evidence of resistance mechanisms to ALK inhibitors are scarce in ALK-dependent tumors other than lung cancer, biological and clinical specificities can be recognized. The relevant results obtained in NSCLC should serve as an example for other ALK-driven diseases.

Purpose: In the literature there are conflicting data about the treatment efficacy of anaplastic lymphoma kinase (ALK) translocation positive non-small cell lung cancer (NSCLC) patients according to ALK fusion variants. We aimed to study the impact of ALK fusion variants on the survival of first-line crizotinib-treated NSCLC patients. Methods: 101 locally advanced or metastatic ALK positive NSCLC patients treated with first-line crizotinib between January 2013 and December 2019 were retrospectively evaluated. We studied ALK fusion variants in 38 of those patients with adequate tumor tissue with reverse transcription polymerase chain reaction (RT PCR). Patients having ALK fusion variant 1 (v1) and non-variant 1 (non-v1) were compared for survival and response to crizotinib. Results: Median age was 52.5 years (range 35-74), and 22 of 38 patients were male (57.9%). EML-4 ALK v1 was seen in 26 patients (68.4%) and 12 were non-v1 (variant 3a/b in 6, and non-EML-4 ALK variants in 6 patients). Objective response rate was 60.5% in all patients, whereas it was 61.5% in v1 and 58.3% in non-v1 group. Median progression-free survival (PFS) and overall survival (OS) were similar. Median PFS was 13.1 months in v1, and 12.4 months in non-v1 (p=0.232). Median OS was 23.2 months in v1, and 19.4 months non-v1 (p=0.493). Conclusion: ALK v1 and non-v1 patients had the same OS and PFS after first-line crizotinib treatment, however there was a trend for v1 group for better OS. © 2021 Zerbinis Publications. All rights reserved., The study was supported by Turkish Oncology Group.

Purpose Ceritinib is an ALK receptor tyrosine kinase inhibitor approved as first- and second-line treatment in adult patients with ALK + metastatic non-small cell lung cancer (NSCLC). The study investigated the drug–drug interaction (DDI) potential of ceritinib when coadministered with midazolam and warfarin as probe substrates for CYP3A and CYP2C9 activity, respectively. Methods This was a phase I, multicenter, open-label, single sequence, crossover DDI study in 33 adult patients with ALK + NSCLC or other advanced tumors. A single dose of a cocktail consisting of midazolam and warfarin was administered with and without concomitant administration of ceritinib. The primary objective was to evaluate the pharmacokinetics of midazolam and warfarin. Secondary objectives included pharmacokinetics, safety, tolerability, overall response rate (ORR), and duration of response (DOR) of ceritinib 750 mg once daily. Results Ceritinib inhibited CYP3A-mediated metabolism of midazolam, resulting in a markedly increased AUC (geometric mean ratio [90% confidence interval]) by 5.4-fold (4.6, 6.3). Ceritinib also led to an increase in the AUC of S-warfarin by 54% (36%, 75%). The pharmacokinetics and safety profile of ceritinib in this study are consistent with previous reports and no new safety signals were reported. Among the 19 patients with NSCLC, efficacy (ORR: 42.1% and DCR: 63.2%) was similar to that reported previously in studies of pretreated patients with ALK + NSCLC. Conclusion Ceritinib is a strong CYP3A inhibitor and a weak CYP2C9 inhibitor. These findings should be reflected as actionable clinical recommendations in the prescribing information for ceritinib with regards to concomitant medications whose pharmacokinetics may be altered by ceritinib.

Drug-induced interstitial lung disease (DI-ILD) is a rare adverse event associated with targeted therapies that inhibit the anaplastic lymphoma kinase (ALK) protein. Although newer-generation ALK inhibitors such as alectinib significantly improve survival in metastatic ALK-rearranged non-small cell lung cancer (NSCLC), the risk of DI-ILD is similar to that of earlier-generation therapies. Lorlatinib is a third-generation ALK inhibitor that is active in patients with metastatic NSCLC whose tumors have developed secondary resistance to alectinib. While it is associated with low rates of DI-ILD in initial phase 1/2 clinical trials, the safety of lorlatinib in patients with a history of DI-ILD has not been well-described. In this case series, we therefore report two patients with metastatic ALK-rearranged NSCLC who each tolerated lorlatinib following recovery from alectinib-related DI-ILD. Both cases were notable for the acute onset of dyspnea, hypoxia, and diffuse ground-glass opacities within one month of initiating alectinib. With no alternative etiology of pneumonitis identified, both patients were treated empirically for grade 3 DI-ILD with corticosteroids and discontinuation of alectinib. Following rapid clinical recovery and eventual radiographic resolution of opacities, each patient was started on lorlatinib at the time of cancer progression, with neither person developing symptoms or radiographic findings consistent with recurrent DI-ILD. In the following series, we describe these two cases in greater detail and discuss their significance within the context of the prior literature. While further descriptions are needed, our experience suggests that lorlatinib may be a safe therapeutic option in some patients who have recovered from DI-ILD.

Anaplastic lymphoma kinase (ALK) aberration is related to high-risk neuroblastomas and is an important therapeutic target. As acquired resistance to ALK tyrosine kinase inhibitors is inevitable, novel anti-ALK drug development is necessary in order to overcome potential drug resistance against ATP-competitive kinase inhibitors. In this study, to overcome ALK inhibitor resistance, we examined the growth inhibition effects of newly developed ALK-targeting pyrrole-imidazole polyamide CCC-003, which was designed to directly bind and alkylate DNA within the F1174L-mutated ALK gene. CCC-003 suppressed cell proliferation in ALK-mutated neuroblastoma cells. The expression of total and phosphorylated ALK was downregulated by CCC-003 treatment but not by treatment with a mismatch polyamide without any binding motif within the ALK gene region. CCC-003 preferentially bound to the DNA sequence with the F1174L mutation and significantly suppressed tumor progression in a human neuroblastoma xenograft mouse model. Our data suggest that the specific binding of CCC-003 to mutated DNA within the ALK gene exerts its anti-tumor activity through a mode of action that is distinct from those of other ALK inhibitors. In summary, our current study provides evidence for the potential of pyrrole-imidazole polyamide ALK inhibitor CCC-003 for the treatment of neuroblastoma thus offering a possible solution to the problem of tyrosine kinase inhibitor resistance.