Anaplastic Lymphoma Kinase Fusion Gene Research Articles

Chinese expert consensus on iruplinalkib for the treatment of locally advanced or metastatic ALK-positive non-small cell lung cancer (2024 edition)

Anaplastic lymphoma kinase (ALK) fusion gene is one of the most common driver gene in non-small cell lung cancer (NSCLC). Epidemiological data showed that ALK gene fusion is detected in 9.06% of Chinese advanced NSCLC patients. ALK-tyrosine kinase inhibitors (TKIs) have become the standard treatment for advanced NSCLC patients with ALK gene fusion. Seven different ALK-TKIs have been approved by the National Medical Products Administration (NMPA) of China, including crizotinib, ceritinib, alectinib, ensartinib, brigatinib, lorlatinib, and iruplinalkib. Iruplinalkib is a novel new-generation ALK-TKI independently developed in China. On June 27, 2023, the NMPA approved iruplinalkib for the treatment of locally advanced or metastatic ALK-positive NSCLC patients whose disease has progressed after previous treatment with crizotinib or who are intolerant to crizotinib. On January 16, 2024, the NMPA approved iruplinalkib for the first-line treatment of locally advanced or metastatic ALK-positive NSCLC patients. In order to better understand the efficacy and safety of iruplinalkib, and facilitate more rationally clinical application of iruplinalkib, the Medical Oncology Branch of China International Exchange and Promotive Association for Medical and Health Care and the Chinese Association for Clinical Oncologists co-organized experts to compile the "Chinese expert consensus on iruplinalkib for the treatment of locally advanced or metastatic ALK-positive non-small cell lung cancer (2024 edition)".

Environment-Sensitive Fluorescent Probe Enables Assessment of Anaplastic Lymphoma Kinase Activity in Nonsmall Cell Lung Cancer.

Anaplastic lymphoma kinase (ALK) rearrangements have been identified as key oncogenic drivers of a subset of nonsmall cell lung cancer (NSCLC). The final chimeric protein of the fusion gene can be constitutively activated, which accounts for the growth and proliferation of ALK-rearranged tumors and thus strongly associates with cancer invasion and metastasis. Diagnostic tools enabling the visualization of ALK activity in a structure-function-based approach are highly desirable to determine ALK status and guide ALK tyrosine kinase inhibitor (ALK-TKI) treatment making. Here, we describe the design, synthesis, and application of a new environment-sensitive fluorescent probe HX16 by introducing an environment-sensitive fluorophore 4-sulfonamidebenzoxadiazole to visualize ALK activity in living cancer cells and tumor tissue slices (mouse model and human biopsy sample). HX16 is a multifunctional chemical tool based on the pharmacophore of ALK-TKI (ceritinib) and can specifically target the kinase domain of ALK with a high sensitivity. Using flow cytometry and confocal microscopy, HX16 enables visualization of ALK activity in various cancer cells with distinct ALK fusion genes, as well as xenograft mouse models. Importantly, HX16 was also applied to visualize ALK activity in a tumor biopsy from a NSCLC patient with ALK-echinoderm microtubule-associated protein-like-4 fusion gene for prediction of ALK-TKI sensitivity. These results demonstrate that strategically designed ALK-TKI-based probe allows the assessment of ALK activity in tumor tissues and hold promise as a useful diagnostic tool in predicting ALK-TKI therapy response.

Case report: ALK-positive histiocytosis presented as bilateral synchronous breast masses with long-term remission on crizotinib

ALK-positive histiocytosis (APH) is a rare type of histiocytic neoplasm with characteristic ALK (Anaplastic Lymphoma Kinase) gene translocation and fusion, with only 27 reported cases in the literature. In this study, we report the first case of synchronous bilateral breast involvement of ALK-positive histiocytosis on initial presentation in a 46-year-old Hispanic woman. APH was diagnosed by the confirmation of clonal histiocyte proliferation with ALK overexpression on IHC and the presence of KIF5B-ALK gene fusion from her breast and lung biopsies. The patient in our study is currently under complete and long-term remission with crizotinib treatment (an ALK inhibitor). This report expands on the clinical manifestation of APH, emphasizes the importance of ALK detection in histiocytic diseases, and provides the efficacy and long-term prognosis of the ALK inhibitor therapy for APH.

A Combination of Alectinib and DNA-Demethylating Agents Synergistically Inhibits Anaplastic-Lymphoma-Kinase-Positive Anaplastic Large-Cell Lymphoma Cell Proliferation.

The recent evolution of molecular targeted therapy has improved clinical outcomes in several human malignancies. The translocation of anaplastic lymphoma kinase (ALK) was originally identified in anaplastic large-cell lymphoma (ALCL) and subsequently in non-small cell lung carcinoma (NSCLC). Since ALK fusion gene products act as a driver of carcinogenesis in both ALCL and NSCLC, several ALK tyrosine kinase inhibitors (TKIs) have been developed. Crizotinib and alectinib are first- and second-generation ALK TKIs, respectively, approved for the treatment of ALK-positive ALCL (ALK+ ALCL) and ALK+ NSCLC. Although most ALK+ NSCLC patients respond to crizotinib and alectinib, they generally relapse after several years of treatment. We previously found that DNA-demethylating agents enhanced the efficacy of ABL TKIs in chronic myeloid leukemia cells. Moreover, aberrant DNA methylation has also been observed in ALCL cells. Thus, to improve the clinical outcomes of ALK+ ALCL therapy, we investigated the synergistic efficacy of the combination of alectinib and the DNA-demethylating agent azacytidine, decitabine, or OR-2100 (an orally bioavailable decitabine derivative). As expected, the combination of alectinib and DNA-demethylating agents synergistically suppressed ALK+ ALCL cell proliferation, concomitant with DNA hypomethylation and a reduction in STAT3 (a downstream target of ALK fusion proteins) phosphorylation. The combination of alectinib and OR-2100 markedly altered gene expression in ALCL cells, including that of genes implicated in apoptotic signaling, which possibly contributed to the synergistic anti-ALCL effects of this drug combination. Therefore, alectinib and OR-2100 combination therapy has the potential to improve the outcomes of patients with ALK+ ALCL.

Alk-rearranged lung adenocarcinoma: From molecular genetics to therapeutic targeting.

Anaplastic Lymphoma Kinase (ALK) is a potent oncogenic driver of lung adenocarcinoma (LUAD). ALK is constitutively activated by gene fusion events between the ALK and other gene fusion partners in about 2-3% of LUADs, characterized by few other gene alterations. ALK-fusions are a druggable target through potent pharmacological inhibitors of tyrosine kinase activity. Thus, several ALK-TKIs (Tyrosine Kinase Inhibitors) of first-, second- and third-generation have been developed that improved the outcomes of ALK-rearranged LUADs when used as first- or second-line agents. However, resistance mechanisms greatly limit the durability of the therapeutic effects elicited by these TKIs. The molecular mechanisms responsible for these resistance mechanisms have been in part elucidated, but overcoming acquired resistance to ALK-derived therapy remains a great challenge. Some new therapeutic strategies under investigation aim to induce long-term remission in ALK-fusion positive LUADs.

Mutation status analysis of 58 patients with advanced ALK fusion gene positive non small cell lung cancer

PurposeTo analyze the characteristics and prognostic values of Anaplastic Lymphoma Kinase (ALK) fusion gene partner, gene subtype and abundance in tumor tissues of advanced Non Small Cell Lung Cancer (NSCLC) patients with positive ALK fusion gene and to explore the best treatment mode of ALK-Tyrosine Kinase Inhibitors(TKIs).MethodsCases of advanced NSCLC patients with ALK positive confirmed by both Next Generation Sequencing (NGS) and immunohistochemistry were retrospectively collected. The relationships of Overall Survival (OS)/Progression Free Survival (PFS) between different mutation subtypes, mutation abundance, clinicopathological features were analyzed. OS/PFS between different treatment mode of ALK inhibitors were compared.ResultsFifty-eight patients were enrolled. There were diverse fusion partners. Five subtypes of Echinoderm Microtubule-associated protein-Like 4 gene (EML4)-ALK fusion mutation were detected: V1,V2,V3,V5 and V7. The mutation abundance ranged from 0.13 to 27.77%, with a median of 5.34%. The abundance of V2 and V5 was higher than V1 and V3 respectively. There was no difference in OS between the low abundance group(≤ 5.34%) and the high abundance group(>5.34%) (P = 0.434). PFS of second-generation ALK inhibitors as first-line treatment was longer than that of Crizotinib as first-line (P<0.001). Never smokers had longer OS than current smokers(P = 0.001).ConclusionsThere are differences in abundance between different fusion partners and subtypes in advanced NSCLC with positive ALK. OS is not associated with subtypes, mutation abundance and first line treatment option of either generation of ALK inhibitors. Smoking is a poor prognostic factor.

Inflammatory myofibroblastic tumors: recent progress and future of targeted therapy.

An inflammatory myofibroblastic tumor is a rare component of bone and soft-tissue sarcomas that has distinct pathological features as a lymphoplasmacytic inflammatory infiltrate. As is the case for other non-small round cell sarcomas, surgical resection remains the standard treatment strategy for inflammatory myofibroblastic tumors, but recurrence is possible. Concerning systemic therapy, the available data for conventional chemotherapy (such as those of doxorubicin-based regimens) are limited, and case reports of anti-inflammatory inflammatory myofibroblastic tumor treatments describe some degree of symptom relief and efficacy against tumor progression. However, as more information about cancer genomics accumulates, the potential for molecularly targeted therapies for inflammatory myofibroblastic tumors has become more promising. Approximately half of inflammatory myofibroblastic tumors harbor anaplastic lymphoma kinase (ALK) fusion genes, and the other half could have potentially targetable fusion genes or mutations such as ROS1, NTRK and RET; case reports demonstrating the clinical efficacy of treatments targeted to inflammatory myofibroblastic tumor have been published, as have several prospective clinical trials. Few drugs are approved for the treatment of inflammatory myofibroblastic tumor, and most of them were approved for tumor-agnostic indications. Drugs that could be used for pediatric indications and dosing in inflammatory myofibroblastic tumor have also not been established. To provide effective targeted therapy for rare diseases such as inflammatory myofibroblastic tumor, it is necessary to obtain clinical evidence by designing and performing clinical trials and to find a path toward regulatory approval.

Genetic profiling of circulating cell-free DNA from cerebrospinal fluid and paired plasma in ALK-positive lung cancer with brain metastases.

This study used next-generation sequencing (NGS) to investigate the genetic profiles in cerebrospinal fluid (CSF), plasma, and tumor tissue to explore alternative detection methods for anaplastic lymphoma kinase (ALK) rearrangement status and potential resistance mechanisms to ALK inhibitors. From January 2016 to January 2021, 19 non-small cell lung cancer (NSCLC) patients with brain metastases (BMs) and ALK-positive primary tumors were enrolled at Beijing Chest Hospital. CSF, plasma, and primary tumor samples from patients with BMs of NSCLC were tested using NGS with a 168-gene panel. The intracranial response and prognosis were also investigated. The study included 19 patients, seven females and 12 males, aged between 29 and 68 (median age 44). CSF cytology was negative in all cases. NGS results showed ALK fusion genes detected in 26.3% (5/19) of CSF cfDNA samples, 78.9% (15/19) of plasma samples, and 89.5% (17/19) of tumor samples from ALK-positive patients. ALK-positive CSF samples had significantly higher allele fractions in CSF cfDNA compared with the other two sample types. In five patients with ALK-positive in CSF, after receiving ALK inhibitors ± local treatment, one case achieved an intracranial complete response, and two had an intracranial partial response. In CSF samples, the intracranial median progression-free survival was 8.0 and 18.0 months for ALK-positive (n = 5) and ALK-negative (n = 14), respectively (p = 0.077). CSF may serve as a liquid biopsy for ALK-positive lung cancer with BMs by detecting cfDNA within CSF to characterize driver and resistant genes.

ZYY-B-2, a novel ALK inhibitor, overcomes resistance to ceritinib by inhibiting P-gp function and induces apoptosis through mitochondrial pathway in ceritinib-resistant H2228 cells

Targeting the Echinoderm microtubule-associated protein-like 4 and anaplastic lymphoma kinase (EML4-ALK) fusion gene is a promising therapeutic strategy for non–small-cell lung cancer (NSCLC) patients. With the advent of the first- and second-generation ALK inhibitors, the mortality rate of lung cancer has shown a downward trend, but almost inevitably, patients will eventually develop resistance, which severely limits the clinical application. Hence, developing new ALK inhibitors which can overcome resistance is essential.Here, we synthesized a novel ALK inhibitor 1-[4-[[5-Chloro-4-[[2-[(1-methylethyl)sulfonyl]phenyl]amino]-2-pyrimidinyl]amino]-3-methoxyphenyl]-3-[2-(4-methyl-1-piperazinyl)-2-oxoethyl]-2-imidazolidinone (ZYY-B-2) based on the structure of the second-generation ALK inhibitor ceritinib. ZYY-B-2 exhibited impressive anti-proliferative effect in the EML4-ALK positive H2228 cells and ceritinib-resistant H2228 (H2228/Cer) cells. Meanwhile, ZYY-B-2 inhibited the activation of p-ALK in a concentration-dependent manner, and inactivated its downstream target proteins p-AKT and p-ERK to inhibit cell proliferation. Subsequently, we found that ZYY-B-2 blocked H2228 cells and H2228/Cer cells in G0/G1 phase and induced cells to undergo apoptosis through the mitochondrial pathway. The ability of its anti-proliferation and pro-apoptosis was significantly stronger than the second generation ALK inhibitor ceritinib. In addition, high expression of P-gp was found in H2228/Cer cells compared with H2228 cells. ZYY-B-2 could inhibit the expression of P-gp in a dose-dependent manner to overcome ceritinib resistance, and the suppression effect of ZYY-B-2 on P-gp might be related to its inhibition of PI3K/AKT signaling pathway.In summary, ZYY-B-2, a promising ALK inhibitor, shows potent activity against ceritinib-resistant cells, which provides experimental and theoretical basis for the further development of new ALK inhibitors.

Molecular Anatomy of the EML4-ALK Fusion Protein for the Development of Novel Anticancer Drugs.

The EML4 (echinoderm microtubule-associated protein-like 4)-ALK (anaplastic lymphoma kinase) fusion gene in non-small-cell lung cancer (NSCLC) was first identified in 2007. As the EML4-ALK fusion protein promotes carcinogenesis in lung cells, much attention has been paid to it, leading to the development of therapies for patients with NSCLC. These therapies include ALK tyrosine kinase inhibitors and heat shock protein 90 inhibitors. However, detailed information on the entire structure and function of the EML4-ALK protein remains deficient, and there are many obstacles to overcome in the development of novel anticancer agents. In this review, we describe the respective partial structures of EML4 and ALK that are known to date. In addition to their structures, noteworthy structural features and launched inhibitors of the EML4-ALK protein are summarized. Furthermore, based on the structural features and inhibitor-binding modes, we discuss strategies for the development of novel inhibitors targeting the EML4-ALK protein.

Identification and therapeutic evaluation of ALK rearrangements in non-small-cell lung cancer.

This study aimed to present a comprehensive assessment of anaplastic lymphoma kinase (ALK) rearrangements evaluated by DNA/RNA‐based next‐generation sequencing (NGS) and Ventana immunohistochemistry (IHC) in patients with non‐small‐cell lung cancer (NSCLC) and to evaluate the therapeutic outcomes of ALK tyrosine kinase inhibitor (TKI) treatment. We investigated ALK gene fusions in 14,894 patients with NSCLC using Ventana IHC and NGS, including 12,533 cases detected via DNA‐based NGS and 2,361 cases using RNA‐based NGS. The overall percentage agreement (OPA), positive percentage agreement (PPA), and negative percentage agreement (NPA) were calculated when comparing the results between NGS and IHC. The therapeutic responses to ALK‐TKIs were also evaluated. In total, 3.50% (439/12,533) of specimens were NGS ALK‐positive (NGS‐p) in the DNA‐based NGS cohort and 3.63% (455/12,533) were IHC ALK‐positive (IHC‐p). The OPA of NGS was 99.60%, whereas its PPA and NPA were 92.75 and 99.86%, respectively. In the adenocarcinoma (ADC) subcohort, the PPA was 95.69%. In the RNA‐based NGS cohort, 2.20% (52/2,361) of specimens were NGS‐p and 2.63% (62/2,361) were IHC‐p. The OPA of NGS was 99.49%; its PPA and NPA were 82.26 and 99.96%, respectively. Thirteen patients with discordant results received ALK‐TKI treatment. In the seven NGS‐p/IHC‐negative (IHC‐n) patients, the overall response rate (ORR) was 85.4% (6/7) and the disease control rate (DCR) was 100%. In the six NGS‐negative/IHC‐p patients, the ORR was 66.7% (4/6) and the DCR was 100%. In summary, a high concordance of ALK gene fusion detected via NGS and IHC was observed in this study. DNA‐based NGS had a higher OPA, PPA, and PPA in the ADC subcohort, whereas RNA‐based NGS had a higher NPA. Overall, the results suggest that the combination of NGS and IHC can improve the accuracy of ALK fusion detection; hence, a result determination algorithm for clinical detection of ALK gene fusion was also proposed.

Immune-related molecular characteristics of Chinese lung cancer patients with different types of ALK rearrangement.

e21144 Background: Anaplastic lymphoma kinase ( ALK) gene rearrangements are found in approximately 5% of non-small-cell lung cancer (NSCLC). Since ALK-TKIs have been developed for patients with ALK-positive advanced NSCLC, it remained 20%-30% non-responders, thus other treatment strategies would be explored to prolong overall survival. Previous studies have shown that ALK rearrangement can upregulate PD-L1 expression via signaling pathways such as ERK/AKT, and the high expression of PD-L1 negatively affects the efficacy of crizotinib and would be more suitable for immunotherapy. However, only a few immunotherapy clinical studies recruited tiny amounts of ALK rearrangement NSCLC, making it difficult to investigate the relationship between this oncogenic gene alteration and therapeutic effects. Herein, we described the immune-related molecular characteristics of ALK-rearranged patients in a large group of Chinese lung cancer, hoping to provide more precise treatment strategies. Methods: A total of 1410 lung cancer patients harboring ALK rearrangement were included in this analysis. Using wide NGS panel testing, we elucidated ALK gene fusions, intergenic region fusions ( ALK-IR) as well as other genomic alterations and TMB levels in each patients’ tumor specimen. PD-L1 expression level was evaluated by the tumor positive score (TPS). Results: Over two hundreds of types of ALK rearrangement were identified. The whole population was divided into six subgroups according to types of ALK rearrangements: 3'- ALK-only (71.1%), 5'-non- EML4-ALK-only (1.0%), multi- ALK fusion (14.2%), single ALK-IR (3.2%), multi-3'- ALK-IR (10.1%), and multi-5'- ALK-IR (0.5%). The overall PD-L1 expression level was high in ALK rearrangement lung cancer, of which the proportion of patients with TPS ≥ 50% was 19.5%, and the TPS ≥ 50% proportion in the single ALK-IR subgroup was the highest (28.6%) among all subgroups. We then evaluated the distribution of TMB levels and found the median TMB level was higher than 30% lung cancer. Of note, the single ALK-IR subgroup also present a trend of higher TMB level among all subgroups. We also observed more DNA damage response (DDR) gene mutations (median number: 2, range: 1-22) in the single ALK-IR subgroup. Somatic copy number amplifications (SCNAs) were detected in 195 (13.8%) ALK -rearranged lung cancer patients, and the proportion of SCNAs in each subgroup was similar. The most frequently high-level amplified genes were MYC (23.9%) and EGFR (10.9%). Furthermore, the 3'- ALK-only subgroup mainly occurred low-level SCNAs (68.5%), whereas the multi- ALK fusion and multi-3'- ALK-IR subgroups were more likely to harbor high and moderate level SCNAs. Conclusions: Our findings demonstrated that lung cancer with ALK-IR might have active immune microenvironment, indicating it’s a subset of ALK-rearranged patients who would benefit from immunotherapy.

Impact of compound mutations I1171N + F1174I and I1171N + L1198H on the structure of ALK in NSCLC pathogenesis: atomistic insights

Anaplastic lymphoma kinase (ALK) fusion genes are found in 3%–5% of non-small cell lung cancers (NSCLCs). NSCLC is the most common type of lung cancer, accounting for 84% of all lung cancer diagnoses. Available treatment options for ALK-positive NSCLCs involve the use of ALK tyrosine kinase inhibitors (ALK-TKIs) which have shown to be effective with a high response rate. Nonetheless, the emergence of multiple compound mutations such as I1171N + F1174I or I1171N + L1198H has been reported to cause resistance to all approved ALK-TKIs. However, the underlying molecular mechanisms surrounding the impact of these compound mutants remain poorly understood. Hence, we performed molecular dynamics simulations to characterize the structural effects and functional implications of these compound mutations. Findings revealed a destabilizing effect on ALK by mutants as compared to the wild-type ALK structure. Also, further insights revealed a lower root-mean-squared fluctuation, radius of gyration, and solvent-accessible surface area values of I1171N + F1174I and I1171N + L1198H ALK compound mutations suggesting that the mutants have a more compact structure and a smaller surface area than the wild-type protein. The mutants also distorted the activation loop residues (Tyr1278, Tyr1282, and Tyr1283) in the ALK structure, which further identify them as possible disruptors of phosphorylation. In contrast to wild conformation, the mutant conformations exhibited a reduced node degree in their residue interaction networks. Collectively, our findings provide deeper insights into the deleterious effects of I1171N + F1174I and I1171N + L1198H ALK compound mutations, which may contribute to NSCLC pathogenesis. Communicated by Ramaswamy H. Sarma

Histopathologic and Molecular Characterization of Uterine Leiomyoma-like Inflammatory Myofibroblastic Tumor: Comparison to Molecular Subtypes of Uterine Leiomyoma.

Uterine leiomyoma (UL) is a common benign neoplasm which can sometimes be difficult to differentiate from the uterine inflammatory myofibroblastic tumor (IMT) based on morphology alone. IMT is a myofibroblastic/fibroblastic neoplasm which has typically been considered to be rare in the uterus. Its clinical behavior is usually indolent although aggressive variants exist. The majority of IMTs harbor genomic rearrangement of anaplastic lymphoma kinase ( ALK ), while ALK fusion has not been thus far detected in ULs. We analyzed 2263 ULs of which 9 (0.4%) had tyrosine-kinase activation. Seven of the samples were ALK immunopositive: 6 had an ALK fusion gene and 1 overexpressed an ALK transcript skipping exons 2 to 3, Moreover, 1 sample had a RET , and 1 a PDGFRB fusion gene. While no recurrent somatic mutations were found, 1 patient had an ALK germline mutation. Seven tumors showed leiomyoma-like morphology, 1 tumor had slightly loose, and 1 fibrous growth pattern. Six tumors had mild to moderate lymphocyte infiltration, while no immune cell infiltration was detected in 3 cases. None of the tumors showed aggressive behavior. Except for strong ALK positivity (7/9 tumors) the protein expression profile of the tumors was identical to ULs and distinct from other mesenchymal uterine tumors. In gene expression level, these tumors and the known UL subclasses did not separate perfectly. However, vitamin C metabolism and epithelial-mesenchymal transition pathways were uniquely enriched in these lesions. The overall similarity of the analyzed tumors to UL raises the question whether an UL diagnosis would be more proper for a subset of uterine IMTs.

Clinical characteristics of non-small cell lung cancer patients with EGFR mutations and ALK&ROS1 fusions.

ObjectiveTo study the relationship between clinical characteristics and anaplastic lymphoma kinase (ALK) fusions, c‐ros oncogene 1, receptor tyrosine kinase (ROS1) gene fusions, and epidermic growth factor receptor (EGFR) mutations in non‐small cell lung cancer (NSCLC) patients to distinguish these different types.MethodsBoth ALK, ROS1 gene rearrangements and EGFR mutations testing were performed. The clinical characteristics and associated pulmonary abnormalities were investigated.ResultsFour hundred fifty‐three NSCLC patients were included for analysis. One hundred seventy (37.5%), 32 (7.1%), and 9 cases (2.0%) with EGFR mutations, ALK gene fusions, and ROS1 gene fusions were identified, respectively. The EGFR‐positive and ALK&ROS1‐positive were more common in female (χ 2 = 61.934, P < 0.001 and χ 2 = 28.152, P < 0.001), non‐smoking (χ 2 = 59.315, P < 0.001 and χ 2 = 11.080, P = 0.001), and adenocarcinoma (χ 2 = 44.864, P < 0.001 and χ 2 = 12.318, P = 0.002) patients; proportion of patients with emphysema was lower (χ 2 = 35.494, P < 0.001 and χ 2 = 15.770, P < 0.001) than the wild‐type patients. The results of logistic regression analysis indicated that female (adjusted odds ratio [OR] 1.834, 95% confidence interval [CI] 1.069–3.144, P = 0.028), non‐smoking (adjusted OR 2.504, 95% CI 1.456–4.306, P = 0.001), lung adenocarcinoma (adjusted OR 4.512, 95% CI 2.465–8.260, P < 0.001), stage III–IV (adjusted OR 2.232, 95% CI 1.066–4.676, P = 0.033), and no symptoms of emphysema (adjusted OR 2.139, 95% CI 1.221–3.747, P = 0.008) were independent variables associated with EGFR mutations. Young (adjusted OR 3.947, 95% CI 1.873–8.314, P < 0.001) and lung adenocarcinoma (adjusted OR 2.950, 95% CI 0.998–8.719, P = 0.050) were associated with ALK/ROS1 fusions.Conclusions EGFR mutations were more likely to occur in non‐smoking, stage III–IV, and female patients with lung adenocarcinoma, whereas ALK&ROS1 gene fusions were more likely to occur in young patients with lung adenocarcinoma. Emphysema was less common in patients with EGFR mutations.

EML4-ALK G1202R mutation induces EMT and confers resistance to ceritinib in NSCLC cells via activation of STAT3/Slug signaling

The echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion gene occurs in approximately 5% of non-small-cell lung cancers (NSCLCs). The development of ALK tyrosine kinase inhibitors (ALK-TKIs) is a major advance in treating NSCLC with the ALK fusion gene. Nevertheless, acquired resistance to ALK-TKIs ultimately limits their use. A prevalent mechanism of drug resistance in kinases occurs through the mutation of G1202R in ALK. However, the mechanisms underlying G1202R resistance to ceritinib are not fully understood. Here, we demonstrated that the expression of EML4-ALK G1202R mutation in A549 cells induced an epithelial-mesenchymal transition (EMT) phenotype and significantly increased the migration and invasion abilities. These phenomena may be due to the upregulation of signal transducer and activator of transcription 3 (STAT3), accompanied by the elevated expression of Slug in EML4-ALK G1202R mutant cells. Furthermore, the combination of ALK and STAT3 inhibitors restored the sensitivity of EML4-ALK G1202R mutant cells to ceritinib. In conclusion, these data indicate that the EML4-ALK G1202R mutation mediates the EMT phenotype by activating the STAT3/Slug signaling pathway, resulting in resistance to ceritinib, and that the combination of STAT3 and ALK inhibitors may overcome ALK mutation-driven drug resistance in the clinic.

Recommendations from Experts in the Management of Adverse Reactions to ALK Inhibitors (2021 Version)

间变性淋巴瘤激酶（anaplastic lymphoma kinase, ALK）融合基因作为肿瘤驱动基因，对非小细胞肺癌（non-small cell lung cancer, NSCLC）的发生和发展至关重要，而靶向ALK融合基因已成为ALK阳性NSCLC患者的主要治疗手段。第一、二代ALK抑制剂（ALK inhibitor, ALKi）克唑替尼、塞瑞替尼、阿来替尼、恩沙替尼已在中国上市并广泛应用。然而，针对ALKi不良反应尚无统一的管理指导规范，在一定程度上降低甚至限制了ALKi的临床使用及患者获益。本文由四川省肿瘤学会肺癌专业委员会及四川省肿瘤性疾病医疗质量控制中心牵头，针对国内已经获批上市的ALKi，参考国内外临床研究和相关文献，并结合专家实践经验，总结出《ALKi不良反应管理专家建议（2021年版）》，以期为临床提供切实可行的ALKi不良反应的管理策略。

Development of a novel ALK rearrangement screening test for non-small cell lung cancers.

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.

Pharmacophore screening, molecular docking, ADMET prediction and MD simulations for identification of ALK and MEK potential dual inhibitors

Anaplastic lymphoma kinase (ALK) fusion gene is a common driver gene in non-small cell lung cancer (NSCLC). The activation of mitogen-activated protein kinase (MAPK) and other related signaling pathways cause the proliferation of cancer cells. Mitogen-activated protein kinase kinase (MAPKK, also known as MEK) is a member of the ALK-MAPK signaling cascade. Recent studies have found that the drug resistance in ALK-positive NSCLC is highly dependent on the activation of the MAPK pathway, and the combined inhibition of ALK and MEK can delay or even eliminate the resistance. In this work, dual ALK/MEK inhibitors were designed through computer-aided drug design (CADD). Ten million molecules from ZINC were screened through pharmacophore models, ADMET prediction and molecular docking. Finally, 35 hit compounds were obtained. Among them, compound 1 has the highest dual inhibitory potential. The results of molecular docking, ADMET prediction and molecular dynamics (MD) simulations show that compound 1 has good potential inhibitory ability to both ALK and MEK, and it also has good druggability. We further modified the structure of compound 1, and two new compounds with significantly improved binding affinity were obtained.

Abstract 1225: LNS8801: A novel, enantiomerically pure, small molecule agonist of the G protein-coupled estrogen receptor (GPER) for the treatment of cancer

Abstract Women have a lower incidence of and improved clinical outcomes for most cancer types, but the mechanistic basis for this sex difference is unknown. We hypothesized that estrogen signaling may be responsible, despite the fact that many cancer types lack classic nuclear estrogen receptors. The GPER agonist G-1, a racemate, has been demonstrated to have anticancer activities by many independent laboratories, though there are some contradictory findings in the literature. We resolved the enantiomers that compose G-1 and determined that LNS8801 is the sole enantiomer responsible for G-1's anticancer effects. Using CRISPR to deplete GPER, we determined that the anticancer activity of LNS8801 is dependent upon GPER expression within the tumor cells. Previously, we determined that the anticancer mechanism of GPER activation is through a rapid proteasomal degradation of c-Myc. The PRISM screen was performed on LNS8801 at the Broad Institute, and it was determined that this compound has universal anticancer activity across multiple cancer types, consistent with our inhouse data. Sensitivity to LNS8801 was highly correlated with expression of c-Myc target genes, which is consistent with the known mechanism of action. Lineage analysis identified two specific cancer types that were uniformly more sensitive to LNS8801 than other cancer types: diffuse large B-cell lymphoma (DLBCL) and anaplastic large cell lymphoma (ALCL). DLBCL has historically been considered to be highly dependent on c-Myc, which is consistent with the known mechanism of action of LNS8801; however, the identification of ALCL was unexpected. Deeper analysis determined that high expression of anaplastic lymphoma kinase (ALK) fusion genes tightly correlated with sensitivity to LNS8801 in ALCL and other ALK+ cancer types. Using non-small cell lung cancer cell lines engineered to express EML4-ALK, we validated that ALK fusion expression results in increased sensitivity to LNS8801 relative to isogenic controls. This work has identified an enantiomerically pure GPER agonist that has potent activity across many cancer types, with c-Myc-dependent and ALK+ cancer types being enriched for heightened sensitivity. Citation Format: Chris Natale, Tina Garyantes, Todd Ridky. LNS8801: A novel, enantiomerically pure, small molecule agonist of the G protein-coupled estrogen receptor (GPER) for the treatment of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1225.