New strategies for treatment of ALK-rearranged non-small cell lung cancers

The identification of oncogenic alterations in subsets of patients with non-small cell lung cancer (NSCLC) is transforming clinical care. Genomic rearrangements in anaplastic lymphoma kinase (ALK) are detected in 3% to 7% of patients with NSCLC. The ALK tyrosine kinase inhibitor crizotinib has demonstrated clinical efficacy in ALK-rearranged NSCLC patients and was recently approved by the U.S. Food and Drug Administration. Crizotinib is currently under additional phase III clinical development as both initial and second-line therapy for advanced ALK-rearranged NSCLC. However, new challenges in the diagnosis and treatment of this subset of NSCLC have emerged, including the need to determine the most effective means of diagnosing ALK-rearranged NSCLC and the emergence of acquired drug resistance to crizotinib. In this review, we discuss current strategies for treatment and diagnosis, as well as the current knowledge about mechanisms of acquired resistance to crizotinib. Finally, we discuss the strategies that are underway to clinically overcome acquired drug resistance.     

Case report: HER2 amplification as a resistance mechanism to crizotinib in NSCLC with MET exon 14 skipping

Patients with non-small cell lung cancer (NSCLC) harboring MET exon 14 skipping can benefit from crizotinib treatment. Currently, the main resistance mechanisms to crizotinib are MET D1228N and Y1230C mutations. We reported a case of a Chinese NSCLC patient with MET exon 14 skipping detected by targeted next-generation sequencing (NGS) achieved clinical and imaging remission after crizotinib treatment. Then, amplification of multiple genes such as erb-b2 receptor tyrosine kinase 2 (HER2) was detected when disease progressed, indicating novel resistance mechanisms to crizotinib. Ultimately the patient died from cancer-related factors. This was the first NSCLC case with MET exon 14 skipping which reported the HER2 gene amplification at the time of progression during crizotinib treatment, indicating that bypass mechanisms contribute to the development of acquired resistance to MET inhibitors.     

The accelerated path of ceritinib: Translating pre-clinical development into clinical efficacy

The discovery of anaplastic lymphoma kinase (ALK)-rearranged non–small-cell lung cancer (NSCLC) in 2007 led to the development and subsequent approval of the ALK inhibitor crizotinib in 2011. However, despite its clinical efficacy, resistance to crizotinib invariably develops. There is now a next generation of ALK inhibitors, including two that have been approved—ceritinib and alectinib—and others that are in development—brigatinib, lorlatinib and X-396. Ceritinib and the other next-generation ALK inhibitors are more potent than crizotinib and can overcome tumor cell resistance mechanisms. Ceritinib gained US Food and Drug Administration approval in 2014 following accelerated review for the treatment of patients with ALK-positive (ALK+) metastatic NSCLC who have progressed on or are intolerant to crizotinib. In pre-clinical studies, it demonstrated more potent inhibition of ALK than crizotinib in enzymatic assays, more durable responses in xenograft models and the ability to potently overcome crizotinib resistance mutations in vitro (including the gatekeeper mutation). There is also evidence for ceritinib penetration across the blood-brain barrier. In clinical trials, ceritinib has demonstrated durable responses and progression-free survival in ALK-inhibitor–pre-treated and –naïve NSCLC patients, including high overall and intracranial response rates in those with central nervous system metastases. Selective gastrointestinal toxicity of ceritinib, such as diarrhea, nausea and vomiting is generally manageable with prophylactic medication and prompt dose reduction or interruption. Future progress in treating ALK+ NSCLC will focus on determining the optimal sequencing of therapies and strategies to overcome acquired resistance, an ongoing challenge in treating ALK-mutation–driven tumors.     

Acquired MET D1228N Mutations Mediate Crizotinib Resistance in Lung Adenocarcinoma with ROS1 Fusion: A Case Report

Patients with non‐small cell lung cancer (NSCLC) containing ROS1 fusions can have a marked response to the ROS1‐targeted tyrosine kinase inhibitors (TKIs), such as crizotinib. Common resistance mechanisms of ROS1‐fusion targeted therapy are acquired mutations in ROS1. Along with the use of next‐generation sequencing in the clinical management of patients with NSCLC during sequential targeted therapy, many mechanisms of acquired resistance have been discovered in patients with activated tyrosine kinase receptors. Besides acquired resistance mutations, bypass mechanisms of resistance to epidermal growth factor receptor (EGFR)‐TKI treatment are common in patients with EGFR mutations. Here we describe a patient with metastatic lung adenocarcinoma with CD74‐ROS1 fusion who initially responded to crizotinib and then developed resistance by the acquired mutation of D1228N in the MET kinase domain, which showed short‐term disease control for cabozantinib.Key Points

• The D1228N point mutation of MET is an acquired mutation for crizotinib resistance.
• The patient obtained short‐term clinical benefit from cabozantinib therapy after resistance to crizotinib.
• The clinical use of next‐generation sequencing could maximize the benefits of precision medicine in patients with cancer.

     

Therapeutic strategies for afatinib‐resistant lung cancer harboring HER2 alterations

Human epidermal growth factor receptor 2 (HER2) plays an important role in the pathogenesis of various cancers. HER2 alterations have been suggested to be a therapeutic target in non‐small‐cell lung cancer (NSCLC), just as in breast and gastric cancers. We previously reported that the pan‐HER inhibitor afatinib could be a useful therapeutic agent as HER2‐targeted therapy for patients with NSCLC harboring HER2 alterations. However, acquired resistance to afatinib was observed in the clinical setting, similar to the case for other HER inhibitors. Thus, elucidation of the mechanisms underlying the development of acquired drug resistance and exploring means to overcome acquired drug resistance are important issues in the treatment of NSCLC. In this study, we experimentally established afatinib‐resistant cell lines from NSCLC cell lines harboring HER2 alterations, and investigated the mechanisms underlying the acquisition of drug resistance. The established cell lines showed several unique afatinib‐resistance mechanisms, including MET amplification, loss of HER2 amplification and gene expression, epithelial‐to‐mesenchymal transition (EMT) and acquisition of cancer stem cell (CSC)‐like features. The afatinib‐resistant cell lines showing MET amplification were sensitive to the combination of afatinib plus crizotinib (a MET inhibitor), both in vitro and in vivo. The resistant cell lines which showed EMT or had acquired CSC‐like features remained sensitive to docetaxel, like the parental cells. These findings may provide clues to countering the resistance to afatinib in NSCLC patients with HER2 alterations.     

ALK inhibition for non-small cell lung cancer: from discovery to therapy in record time

It was only 3 years ago that an acquired translocation of EML4 with ALK leading to the expression of an EML4-ALK oncoprotein in non-small cell lung cancer (NSCLC) was reported. Tumor cells expressing EML4-ALK are "addicted" to its continued function. Now, crizotinib, an oral ALK inhibitor, is demonstrated to provide dramatic clinical benefit with little toxicity in patients having such advanced NSCLC, and a mechanism of clinical resistance to crizotinib is identified. Such therapy "targeted" at oncogenic proteins provides "personalized" medicine and prompts genome-wide mutation analysis of human tumors to find other therapeutic targets.     

Review of the current targeted therapies for non-small-cell lung cancer

The last decade has witnessed the development of oncogene-directed targeted therapies that have significantly changed the treatment of non-small-cell lung cancer (NSCLC). In this paper we review the data demonstrating efficacy of gefitinib, erlotinib, and afatinib, which target the epidermal growth factor receptor (EGFR), and crizotinib which targets anaplastic lymphoma kinase (ALK). We discuss the challenge of acquired resistance to these small-molecular tyrosine kinase inhibitors and review promising agents which may overcome resistance, including the EGFR T790M-targeted agents CO-1686 and AZD9291, and the ALK-targeted agents ceritinib (LDK378), AP26113, alectinib (CH/RO5424802), and others. Emerging therapies directed against other driver oncogenes in NSCLC including ROS1, HER2, and BRAF are covered as well. The identification of specific molecular targets in a significant fraction of NSCLC has led to the personalized deployment of many effective targeted therapies, with more to come.     

Induction of autophagy contributes to crizotinib resistance in ALK-positive lung cancer

Use of the inhibitor of ALK fusion onco-protein, crizotinib (PF02341066), has achieved impressive clinical efficacy in patients with ALK-positive non-small cell lung cancer. Nevertheless, acquired resistance to this drug occurs inevitably in approximately a year, limiting the therapeutic benefits of this novel targeted therapy. In this study, we found that autophagy was induced in crizonitib-resistant lung cancer cells and contributed to drug resistance. We observed that ALK was downregulated in the crizotinib-resistant lung cancer cell line, H3122CR-1, and this was causally associated with autophagy induction. The degree of crizotinib resistance correlated with autophagic activity. Activation of autophagy in crizotinib-resistant H3122CR-1 cells involved alteration of the Akt/mTOR signaling pathway. Furthermore, we demonstrated that chloroquine, an inhibitor of autophagy, could restore sensitivity of H3122CR-1 to crizotinib and enhance its efficacy against drug-resistant lung cancer. Thus, modulating autophagy may be worth exploring as a new strategy to overcome acquired crizonitib resistance in ALK-positive lung cancer.     

Dual ALK and EGFR inhibition targets a mechanism of acquired resistance to the tyrosine kinase inhibitor crizotinib in ALK rearranged lung cancer

Introduction

The multitargeted tyrosine kinase inhibitor (TKI) crizotinib is active against ALK translocated non-small-cell lung cancer (NSCLC); however acquired resistance invariably develops over time. ALK mutations have previously been implicated in only a third of resistant tumors. We sought to evaluate alternative mechanisms of resistance and preclinical strategies to overcome these in a cell line driven by EML4-ALK.

Methods

We selected the NSCLC cell line NCI-H3122 (H3122: EML4-ALK E13;A20) and derived resistant variants that were able to grow in the presence of 1 μM crizotinib. These were analyzed for ALK mutations, sensitivity to crizotinib in combination with other TKIs, and for activation of alternative tyrosine kinases.

Results

All H3122 crizotinib resistant (CR) clones lacked amplification or mutations in the kinase domain of ALK. To evaluate if possible alternative kinases functioned as “bypass” tracks for downstream signaling activation in these resistance cells, we performed of phosho-receptor tyrosine kinase array that demonstrated that CR clones had higher phospho-EGFR signals than H3122 cells before and after exposure to crizotinib. A functional approach of dual ALK TKI (with crizotinib) with combinatory TKI inhibition was used as a secondary screen for possible targets. Crizotinib + erlotinib (reversible EGFR TKI) and crizotinib + afatinib (irreversible EGFR/ERBB2 TKI) were able to inhibit the growth of H3122 CR clones, confirming EGFR activation as a mechanism of resistance. The removal of crizotinib from the culture media re-sensitized CR cells to crizotinib.

Conclusions

We identified activation of EGFR as a mechanism of resistance to crizotinib in preclinical models of ALK translocated NSCLC. If EGFR activation is confirmed as a predominant mechanism of ALK TKI-induced resistance in patient-derived tumors, the use of ALK plus EGFR TKIs could be explored for this important cohort of NSCLCs.     

Overcoming crizotinib resistance in ALK-rearranged NSCLC with the second-generation ALK-inhibitor ceritinib

In up to 5% of non-small cell lung cancer (NSCLC) patients, the EML4-ALK translocation drives tumor progression. Treatment with the ALK inhibitor crizotinib is more effective than standard chemotherapy. However, resistance to crizotinib occurs after approximately 8 months. Ceritinib is the first second-generation ALK inhibitor approved for treatment of crizotinib-resistant NSCLC. Ceritinib inhibits two of the most common ALK-mutants that confer resistance to crizotinib: L1196 M and G1269A. Cells with ALK expression are more sensitive to ceritinib than crizotinib (IC₅₀ 25 nM vs. 150 nM, respectively). Alternative second-generation ALK inhibitors such as Alectinib, Brigatinib and PF-06463922 are currently in development, each affecting different crizotinib-resistant ALK target mutations. Genetic identification of crizotinib-resistant mutants is essential for selecting the optimal treatment strategy in NSCLC patients to overcome resistance and to increase progression-free survival.     

ALK激酶域耐药突变的研究进展及未来应对策略

间变性淋巴瘤激酶（anaplastic lymphoma kinase,ALK）是非小细胞肺癌（non-small cell lung cancer,NSCLC）中常见的致癌驱动基因之一。酪氨酸激酶抑制剂（tyrosine kinase inhibitor,TKI）在ALK融合基因阳性的NSCLC患者中均取得了优异的治疗效果,然而患者最终会对TKI产生耐药性。获得性的分子生物学耐药,如ALK激酶域突变、ALK基因扩增和旁路异常激活等,是影响ALK⁺ NSCLC靶向治疗效果的重要因素。获得性的ALK激酶域耐药突变现已成为关注重点。随着二代基因测序技术（next-generation sequencing,NGS）的不断进步及普及,ALK-TKI的耐药突变谱逐渐清晰,并且获得性耐药可能是动态变化的。首先,第一代、第二代TKI治疗失败后继发ALK激酶域耐药性突变以单点突变为主。约20%的患者在接受克唑替尼治疗失败后出现耐药突变,以L1196M、G1269A、C1156Y和F1174L为主。第二代TKI（包括阿来替尼、塞瑞替尼、布加替尼和恩沙替尼）耐药后点突变的发生率高达50%,且类型更丰富,例如G1202R/del、F1174C/V和I1171T/N/S等。相对于克唑替尼,第二代TKI对ALK激酶具有更高的抑制效果,可覆盖大部分的ALK耐药突变,但G1202R/del除外。研究发现,除G1202R是最常见的第二代TKI耐药性突变外,F1174C/L和I1171N/S/T分别是塞瑞替尼和阿来替尼的主要耐药突变,G1269A和E1210K是恩沙替尼的主要耐药突变位点。其次,第二代TKI耐药后ALK双重突变和“脱靶”比例显著增加。第三代TKI劳拉替尼耐药后几乎均为复合突变,并且耐药程度更高。现已发现I1171N-双重突变及G1202R-双重突变谱,其中,G1202R+L1196M双突变显示出对所有ALK-TKI的高度耐药。此外,序贯多代ALK-TKI治疗进展后,原有耐药位点发生变化,野生型的比列升高,耐药机制可能更为复杂。目前,在克唑替尼耐药后,序贯第二代/第三代TKI可抑制绝大部分耐药突变。而第二代TKI治疗进展后,可通过序贯其他第二代TKI或劳拉替尼达到抑瘤效果。对于顽固性的溶剂前沿区域突变,劳拉替尼对G1202R突变有显著的抑制效果,而对劳拉替尼耐药的L1198F突变及L1198F-双重突变对克唑替尼重新敏感。某些复合突变对第二代TKI敏感,如I1171N+L1196M和I1171N+G1269A突变,大部分复合耐药突变仍未发现有效的抑制剂。有新一代TPX-0131和NVL-655在临床前实验中以表现出优异的抑瘤效果,尤其是能够克服ALK复合耐药突变,但仍需要临床试验的验证。识别ALK-TKI的激酶域耐药突变谱,选择敏感且高效的TKI治疗是近年来的研究热点。本文聚焦于获得性ALK激酶域耐药机制,系统综述了ALK基因背景与激酶域耐药的关系和ALK-TKI激酶域耐药突变谱和治疗策略。同时,肿瘤进展后的重复活检对于识别ALK激酶域突变以及选择最有效的治疗策略至关重要。     

非小细胞肺癌分子靶向治疗中EGFR-TKI的耐药机制研究进展

随着分子靶向治疗药物的发展,以吉非替尼(gefitinib,iressa)和厄洛替尼(erlotinib,tarceva)为代表的表皮生长因子受体酪氨酸激酶抑制剂(epidermal growth factor receptor-tyrosine kinases inhibitors,EGFR-TKIs)在治疗非小细胞肺癌中发挥了重要作用。然而在临床前和临床研究中发现许多患者对此药物存在原发性耐药或获得性耐药,使该类药物的使用受到一定限制。本文就近年来对EGFR-TKIs耐药机制的研究进展进行综述。     

ROS1阳性的非小细胞肺癌的检测及靶向治疗进展

ROS1融合基因是非小细胞肺癌（non-small cell lung cancer，NSCLC）靶向治疗的又一潜力靶点，随着相应靶向药物的使用，ROS1阳性的NSCLC患者生存期明显改善。越来越多针对ROS1融合基因的治疗药物面世，让这类患者有更多的选择。但持续性用药后的获得性耐药问题仍无法避免。本文就ROS1融合基因的检测方法、靶向治疗情况及耐药的机制和策略进行综述。     

Treatment for ALK-mutated non-small-cell lung cancer: a new miracle in the research race

The discovery of anaplastic lymphoma kinase (ALK) rearrangements in a subset of patients with non-small-cell lung cancer (NSCLC) and its potential blockage by specific inhibitors such as crizotinib has been one of the latest advances in the treatment of this disease. In this article, we will review the most important clinical aspects of ALK alterations in NSCLC patients and the pending questions to answer: the most effective means of diagnosing ALK-rearranged NSCLC, and efficacy, toxicity profile and potential mechanisms of resistance to crizotinib.     

ALK‐positive non–small cell lung cancer: Mechanisms of resistance and emerging treatment options

Targeted therapy has emerged as an effective treatment option for certain molecular subsets of advanced stage non–small cell lung cancer (NSCLC). The discovery of the echinoderm microtubule‐associated protein like 4–anaplastic lymphoma kinase (EML4‐ALK) translocation as an oncogenic driver has led to the development of novel therapies with activity in vitro and in the clinic. The first‐in‐class tyrosine kinase inhibitor crizotinib is effective against ALK‐positive NSCLC and is currently used as first‐line or salvage therapy in the setting of advanced disease. However, resistance inevitably develops through a variety of mechanisms, including point mutations affecting the fusion protein, activation of bypass signaling pathways, copy number gain of ALK, and other means. Increased understanding of these pathways is essential for tailoring treatment choices to improve outcomes and minimize toxicities. Potent second‐generation ALK inhibitors currently in trials are producing encouraging results in ALK‐positive NSCLC, even in patients with acquired resistance to crizotinib. The success in identifying the ALK translocations and rapidly developing targeted drugs to exploit it paves the way for a better understanding of NSCLC biology and the quest to provide effective, personalized treatment for lung cancer patients. Cancer 2014;120:2392–2402. © 2014 American Cancer Society.     

ALK融合基因阳性的晚期非小细胞肺癌靶向治疗进展

近十年来，晚期非小细胞肺癌（non-small cell lung cancer，NSCLC）在治疗方面出现重大的模式转变。关键致癌性突变（如驱动基因突变和染色体重排）的存在，使得靶向治疗相比传统的细胞毒性化学疗法显示出更高的敏感性。2007年间变性淋巴瘤激酶（anaplastic lymphoma kinase，ALK）基因与棘皮动物微管相关蛋白样-4（echinoderm microtubule-associated protein-4，EML4）基因融合突变首次在NSCLC患者中被发现。随后研究证实，ALK-EML4融合突变阳性的NSCLC（ALK+NSCLC）显示出对克唑替尼治疗的敏感性。随着后续一系列靶向治疗新药的研发，将ALK+NSCLC靶向治疗推向高潮。本综述回顾ALK+NSCLC的分子生物学发病机制、流行病学特征及检测方法，汇总其抑制剂的重要临床试验结果，并解读ALK+NSCLC抑制剂耐药机制及合并脑转移的最新研究进展。     

Potential therapeutic strategies to overcome acquired resistance to BRAF or MEK inhibitors in BRAF mutant cancers

Recent clinical trials with selective inhibitors of the BRAF and MEK kinases have shown promising results in patients with tumors harboring BRAF V600 mutations. However, as has been observed previously with similarly successful targeted therapies, acquired resistance to these agents is an emerging problem that limits their clinical benefit. Several recent studies from our laboratory and others have investigated the causes of acquired resistance to BRAF and MEK inhibitors, and multiple resistance mechanisms have been identified. Here, we review these mechanisms and suggest that they can be broadly grouped into two main classes: ERK-dependent and ERK-independent. We also propose distinct therapeutic strategies that might be employed to overcome each class of acquired resistance..     

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

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

Resistance to combination BRAF and MEK inhibition in metastatic melanoma: Where to next?

Treatment of BRAF-mutant metastatic melanoma with mitogen-activated protein kinase (MAPK) pathway targeted therapies (BRAF/MEK inhibitors) and immune checkpoint inhibitors has revolutionised management and improved outcomes for patients with advanced stage disease. However, acquired resistance to MAPK inhibitor therapy develops in the majority of patients at approximately 12 months and multiple mechanisms lead to resistance. Understanding the mechanisms of resistance is therefore critical for the development of more effective therapeutic strategies in BRAF-mutant melanoma. Recently, several distinct mechanisms of resistance to BRAF-inhibition have been proposed based on data obtained in experimental melanoma cell models and small series of human tumour samples. These include reactivation of the MAPK pathway resulting in continued extracellular signal-regulated kinase activation and activation of parallel signalling pathways including the PI3K-mTOR (phosphoinositide 3-kinase–mammalian target of rapamycin) pathway. Alterations in how the cells of the immune system respond to melanoma cells treated with targeted therapy may also influence response and progression. In this review, we discuss these mechanisms and identify potential therapeutic strategies to overcome resistance which, in turn, will lead to improved outcomes for patients with metastatic melanoma.     

Detection of ALK and KRAS Mutations in Circulating Tumor DNA of Patients With Advanced ALK-Positive NSCLC With Disease Progression During Crizotinib Treatment

BackgroundIn patients with anaplastic lymphoma kinase (ALK)-positive non–small-cell lung cancer (NSCLC), disease progression occurs after a median of 9 to 10 months of crizotinib treatment. Several mechanisms of resistance have been identified and include ALK mutations and amplification or the activation of bypassing signaling pathways. Rebiopsy in NSCLC patients represents a critical issue and the analysis of circulating cell-free DNA (cfDNA) has a promising role for the identification of resistance mechanisms.Patients and MethodsTwenty patients with advanced ALK-positive NSCLC were enrolled after disease progression during crizotinib treatment; cfDNA was analyzed using digital droplet polymerase chain reaction (BioRad, Hercules, CA) for ALK (p.L1196M, p.G1269A, and p.F1174L) and Kirsten rat sarcoma (KRAS) (codons 12 and 13) mutations.ResultsALK secondary mutations (p.L1196M, p.G1269A, and p.F1174L) were identified in 5 patients; 1 patient had 2 ALK mutations (p.L1196M and p.G1269A). Overall, 10 patients presented KRAS mutations (7 p.G12D, 2 p.G12V, and 1 p.G12C mutations, respectively). In 3 patients KRAS mutations were associated with ALK mutations. cfDNA was monitored during the treatment with second-generation ALK inhibitors and the amount of ALK as well as KRAS mutations decreased along with tumor regression.ConclusionALK and KRAS mutations are associated with acquired resistance to crizotinib in ALK-positive NSCLC. In particular, ALK acquired mutations can be detected in plasma and could represent a promising tumor marker for response monitoring.