Anaplastic Lymphoma Kinase Tyrosine Kinase Research Articles

Crizotinib in non-small cell lung cancer

Chemotherapy and targeted therapy remain the cornerstone of treatment of locally advanced and metastatic non-small cells lung cancer (NSCLC). Given the intrinsic chemoresistance of tumor cells, new treatment options have been developped. The knowledge of the molecular mechanisms of tumor biology, and signal transduction pathways activating cancer cells led to the identification of a new targeted therapy such as Crizotinib. The small molecule Crizotinib is a selective inhibitor of the receptor tyrosine kinase ALK (anaplastic lymphoma kinase) and its oncogenic variants (ALK fusion gene and some mutations of ALK). Phases I and II trials showed the efficacy of Crizotinib in the treatment of locally advanced and metastatic NSCLC expressing ALK. Thereafter, randomized Phase III trial confirmed the significant superiority of Crizotinib versus standard chemotherapy in terms of progression free survival and objective response with good tolerance; therefore, it has been approved by the Food and Drug Administration (FDA) as the standard treatment for locally advanced and metastatic ALK-positive NSCLC.

Testing for ALK rearrangement in lung adenocarcinoma: a multicenter comparison of immunohistochemistry and fluorescent in situ hybridization

Rearrangements of anaplastic lymphoma kinase (ALK) gene in non-small cell lung cancer (NSCLC) define a molecular subgroup of tumors characterized clinically by sensitivity to ALK tyrosine kinase inhibitors such as crizotinib. Although ALK rearrangements may be detected by reverse transcriptase-PCR, immunohistochemistry or fluorescence in situ hybridization (FISH), the optimal clinical strategy for identifying ALK rearrangements in clinical samples remains to be determined. We evaluated immunohistochemistry using three different antibodies (ALK1, 5A4 and D5F3 clones) to detect ALK rearrangements and compared those with FISH. We report the frequency and clinicopathologic features of lung cancers harboring ALK translocations in 594 resected NSCLCs (470 adenocarcinomas; 83 squamous carcinomas, 26 large cell carcinomas and 15 other histological subtypes) using a tissue microarray approach. We identified an ALK gene rearrangement in 7/594 cases (1%) by FISH and all anti-ALK antibodies correctly identified the seven ALK-positive cases (100% sensitivity), although the intensity of staining was weak in some cases. These data indicate that the use of antibodies with high sensitivity and avidity to ALK may provide an effective pre-screening technique to complement the more expensive and labor-intensive approach of ALK FISH testing.

Abstract A284: Anaplastic lymphoma kinase (ALK) inhibitors for cancer treatment.

Abstract Anaplastic lymphoma kinase (ALK), a receptor tyrosine kinase, has been recently elucidated as a potential target for various cancers due to its implications of tumorigenesis by ALK gene mutations, overexpressions, and amplifications. ALK was first identified in 1994 as a part of nucleophosmin NPM-ALK fusion gene in 60% of anaplastic large-cell lymphoma (ALCL). In late 2007, EML4-ALK fusion gene was found in 3∼7% of non-small cell lung cancer (NSCLC), and a kind of ALK fusion genes are found one by one in various cancers such as DLBCL, inflammatory myofiblastic tumor (IMT), plasmacytoma, esophageal cancer, and ovarian cancer. More over mutated ALK is much implicated in neuroblastoma and thyroid carcinoma. Crizotinib (Xalkori) was the first small molecule inhibitor which was approved as a treatment of NSCLC including ALK fusion gene by FDA in 2011. Crizotinib, a potent inhibitor of both c-Met and ALK tyrosine kinases is a 3-benzyloxy-2-aminopyridine derivative derived from c-Met inhibitors and surprisingly its overall clinical benefit was 57 %. However, its clinical efficacy is limited by drug-resistance mutations, particularly the gatekeeper L1196M mutation. Unlike their aminopyridine-based core scaffold, a pyrimidine-based inhibitor has been identified for the ALK-related inhibition and shown a potent activity to EML4-ALK wild type and other mutants including EML4-ALK L1196M. In this paper, we designed and synthesized a new series of pyrimidine derivatives to discover a new ALK inhibitor which is well-matched with crizotinib and LDK378 in docking study. KRCA-0008 has been identified as a highly potent and selective ALK inhibitor with potency profiles (ALK wt, IC50=12 nM). It has excellent activities both ALK mutants (L1196M, C1156Y, F1174L, R1275Q) and BaF3 ALK L1196M cell line. PK profiles were excellent and KRCA-0008 was safe in Ames test, chromosomal aberration assay, micronucleus asssay, and acute toxicity. Also, we observed KRCA-0008 is chemically and metabolically very stable and no CYP and hERG inhibitions. In vivo xenograft mouse model (H3122 NSCLC) model study, KRCA-0008 shows moderate tumor growth inhibition without significant body weight change. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A284. Citation Format: Kwangho Lee, Hyoung Rae Kim, Sung Yun Cho, Hee Jung Jung, Jae Du Ha, Chang-Soo Yun, Pilho Kim, Chi Hoon Park, Chong Ock Lee. Anaplastic lymphoma kinase (ALK) inhibitors for cancer treatment. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A284.

ALK (anaplastic lymphoma kinase, CD246)-specific CARs: new immunotherapeutic agents for the treatment of pediatric solid tumors

Meeting abstracts The identification of unique cell-surface proteins expressed on tumor cells, yet not expressed on normal tissues, has been challenging for pediatric malignancies. The cell surface tyrosine kinase ALK (CD246, anaplastic lymphoma kinase) is a promising target for neuroblastoma in

New Pyrimidine Derivatives possessing ALK Inhibitory Activities

Anaplastic lymphoma kinase (ALK), a family of tyrosine kinases has been recently elucidated as a potential target for various cancers due to its implications of tumorigenesis by ALK gene mutations, overexpressions, and amplifications. ALK was first identified in 1994 as a part of nucleophosmin NPM-ALK fusion gene in 60% of anaplastic large-cell lymphoma (ALCL), in late 2007 EML4-ALK fusion gene was found in 3-7% of non-small cell lung cancer, and a kind of ALK fusion genes are found one by one in various cancers such as DLBCL, inflammatory myofibroblastic tumor (IMT), plasmacytoma, esophageal cancer, renal cell carcinoma, breast cancer, colon cancer, and ovarian cancer. Moreover mutated ALK is much implicated in neuroblastoma and thyroid carcinoma. Crizotinib (Xalkori) was the first small molecule inhibitor which was approved as a treatment of NSCLC including ALK fusion gene by FDA in 2011. Crizotinib, a potent inhibitor of both c-Met and ALK tyrosine kinases is a 3-benzyloxy-2-aminopyridine derivative derived from c-Met inhibitors and surprisingly its overall clinical benefit was 87%. Many pharmaceutical companies have been looking forward to finding better ALK inhibitors and among them, CH-5424802 by Chugai Pharm., LDK378 by Novartis/GNF, and AZD-3463 by Astrazeneca are showing excellent progress. 2-Aminopyrimidine derivatives have been massively studied as various kinase inhibitors such as for ALK, EGFR, Aurora kinase, JAK, CK1, LRR2, etc. Especially, imatinib and nilotinib for BCR-ABL, dasatinib for BCR-ABL/Src/c-Kit/etc., fostamatinib for Syk, and pazopanib for VEGFR2/PDGFR/c-Kit had approved as new chemical entities by US FDA. We designed a new series of pyrimidine derivatives to discover a new ALK inhibitor, and among them 4-{4-(piperazin-1-)yl-2-methoxy-phenyl}amino-2-(methoxy-substitutedphenyl)aminopyrimidines were well-matched with crizotinib and LDK378 in docking study. We synthesized those pyrimidine derivatives and evaluated ALK inhibitory activities. 5-Substituted-2,4-dichloropyrimidine 1 (X=F, Cl) was reacted with 4-(N-acetylpiperazin-1-yl)-2-methoxyaniline 2 in DMF at 80 C overnight to predominantly afford 4-substituted-pyrimidine 3 in good yield and it was reacted with various methoxyanilines in 0.08 M HCl solution of ethoxyethanol at 110 C overnight to afford 5 in moderate to good yields (Scheme 1). In case of 2,4-dichloro-5-trifluoromethylpyrimidine, reaction of 6 with aniline (2) in the presence of base in DMF gave a mixture of 2-substituted and 4-substituted pyrimidines with a trace of 2,4-disubstituted pyrimidine, and the regioisomers could hardly been separated by TLC. Fortunately, 6 reacted with 4-(N-Boc-piperazin-1-yl)-2-methoxyaniline 7 in the presence of diisopropylethylamine as a base at room

RET fusion gene: Translation to personalized lung cancer therapy

Development of lung adenocarcinoma (LADC), the most frequent histological type of lung cancer, depends in many cases on the activation of "driver" oncogenes such as KRAS, epidermal growth factor receptor (EGFR), and anaplastic lymphoma kinase (ALK). Inhibitors that target the EGFR and ALK tyrosine kinases show therapeutic effects against LADCs containing EGFR gene mutations and ALK gene fusions, respectively. Recently, we and others identified the RET fusion gene as a new targetable driver gene in LADC. The RET fusions occur in 1-2% of LADCs. Existing US Food and Drug Administration-approved inhibitors of RET tyrosine kinase show promising therapeutic effects both in vitro and in vivo, as well as in a few patients. Clinical trials are underway to investigate the therapeutic effects of RET tyrosine kinase inhibitors, such as vandetanib (ZD6474) and cabozantinib (XL184), in patients with RET fusion-positive non-small-cell lung cancer.

Mechanistic insight into ALK receptor tyrosine kinase in human cancer biology

The burgeoning field of anaplastic lymphoma kinase (ALK) in cancer encompasses many cancer types, from very rare cancers to the more prevalent non-small-cell lung cancer (NSCLC). The common activation of ALK has led to the use of the ALK tyrosine kinase inhibitor (TKI) crizotinib in a range of patient populations and to the rapid development of second-generation drugs targeting ALK. In this Review, we discuss our current understanding of ALK function in human cancer and the implications for tumour treatment.

Insight into Crizotinib Resistance Mechanisms Caused by Three Mutations in ALK Tyrosine Kinase using Free Energy Calculation Approaches

As a safe and efficacious drug, crizotinib was approved by the U.S. Food and Drug Administration (FDA) in 2011 for the treatment of advanced fusion-type nonsmall-cell lung cancer. Although high response ratio was detected from the patients treated with crizotinib, the cancer has eventually conferred resistance to crizotinib. Several drug resistance mutations have been found in the anaplastic lymphoma kinase (ALK) tyrosine kinase domain as the target for crizotinib, but the drug resistance mechanisms remain unclear. Therefore, in this study, the adaptive biasing force (ABF) method and two-end-state free energy calculation approaches were employed to elucidate the resistance mechanisms of crizotinib induced by the mutations L1152R, G1202R, and S1206Y. The ABF simulation results suggest that the reaction coordinates for the unbinding processes of crizotinib from the binding pockets of the mutated ALKs is different from that of the wild type ALK. The potentials of mean force for the crizotinib unbinding and the binding free energies predicted by the two-end-state free energy calculations are consistent with the experimental data. Our results indicate that the three mutations weaken the binding affinity of crizotinib obviously and lead to drug resistance. The free energy decomposition analysis illustrates the importance of the loss of two important H-bonds in the L1152R and S1206Y mutants on drug resistance. The entropy analysis shows that the entropy term plays a critical role in the substantial change of the conformational entropies of G1202R and L1152R. Our results reveal the mechanisms of drug resistance and provide vital clues for the development of new inhibitors to combat drug resistance.

Molecular Testing Guideline for Selection of Lung Cancer Patients for EGFR and ALK Tyrosine Kinase Inhibitors: Guideline from the College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology

To establish evidence-based recommendations for the molecular analysis of lung cancers that are that are required to guide EGFR- and ALK-directed therapies, addressing which patients and samples should be tested, and when and how testing should be performed. Three cochairs without conflicts of interest were selected, one from each of the 3 sponsoring professional societies: College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology. Writing and advisory panels were constituted from additional experts from these societies. Three unbiased literature searches of electronic databases were performed to capture articles published published from January 2004 through February 2012, yielding 1533 articles whose abstracts were screened to identify 521 pertinent articles that were then reviewed in detail for their relevance to the recommendations. Evidence was formally graded for each recommendation. Initial recommendations were formulated by the cochairs and panel members at a public meeting. Each guideline section was assigned to at least 2 panelists. Drafts were circulated to the writing panel (version 1), advisory panel (version 2), and the public (version 3) before submission (version 4). The 37 guideline items address 14 subjects, including 15 recommendations (evidence grade A/B). The major recommendations are to use testing for EGFR mutations and ALK fusions to guide patient selection for therapy with an epidermal growth factor receptor (EGFR) or anaplastic lymphoma kinase (ALK) inhibitor, respectively, in all patients with advanced-stage adenocarcinoma, regardless of sex, race, smoking history, or other clinical risk factors, and to prioritize EGFR and ALK testing over other molecular predictive tests. As scientific discoveries and clinical practice outpace the completion of randomized clinical trials, evidence-based guidelines developed by expert practitioners are vital for communicating emerging clinical standards. Already, new treatments targeting genetic alterations in other, less common driver oncogenes are being evaluated in lung cancer, and testing for these may be addressed in future versions of these guidelines.

Molecularly targeted approaches herald a new era of non-small-cell lung cancer treatment

The discovery of activating mutations in the epidermal growth-factor receptor (EGFR) gene in 2004 opened a new era of personalized treatment for non-small-cell lung cancer (NSCLC). EGFR mutations are associated with a high sensitivity to EGFR tyrosine kinase inhibitors, such as gefitinib and erlotinib. Treatment with these agents in EGFR-mutant NSCLC patients results in dramatically high response rates and prolonged progression-free survival compared with conventional standard chemotherapy. Subsequently, echinoderm microtubule-associated protein-like 4 (EML4)–anaplastic lymphoma kinase (ALK), a novel driver oncogene, has been found in 2007. Crizotinib, the first clinically available ALK tyrosine kinase inhibitor, appeared more effective compared with standard chemotherapy in NSCLC patients harboring EML4-ALK. The identification of EGFR mutations and ALK rearrangement in NSCLC has further accelerated the shift to personalized treatment based on the appropriate patient selection according to detailed molecular genetic characterization. This review summarizes these genetic biomarker-based approaches to NSCLC, which allow the instigation of individualized therapy to provide the desired clinical outcome.

Molecular testing guideline for selection of lung cancer patients for EGFR and ALK tyrosine kinase inhibitors: guideline from the College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology.

To establish evidence-based recommendations for the molecular analysis of lung cancers that are required to guide EGFR- and ALK-directed therapies, addressing which patients and samples should be tested, and when and how testing should be performed. Three cochairs without conflicts of interest were selected, one from each of the 3 sponsoring professional societies: College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology. Writing and advisory panels were constituted from additional experts from these societies. Three unbiased literature searches of electronic databases were performed to capture articles published from January 2004 through February 2012, yielding 1533 articles whose abstracts were screened to identify 521 pertinent articles that were then reviewed in detail for their relevance to the recommendations. Evidence was formally graded for each recommendation. Initial recommendations were formulated by the cochairs and panel members at a public meeting. Each guideline section was assigned to at least 2 panelists. Drafts were circulated to the writing panel (version 1), advisory panel (version 2), and the public (version 3) before submission (version 4). The 37 guideline items address 14 subjects, including 15 recommendations (evidence grade A/B). The major recommendations are to use testing for EGFR mutations and ALK fusions to guide patient selection for therapy with an epidermal growth factor receptor (EGFR) or anaplastic lymphoma kinase (ALK) inhibitor, respectively, in all patients with advanced-stage adenocarcinoma, regardless of sex, race, smoking history, or other clinical risk factors, and to prioritize EGFR and ALK testing over other molecular predictive tests. As scientific discoveries and clinical practice outpace the completion of randomized clinical trials, evidence-based guidelines developed by expert practitioners are vital for communicating emerging clinical standards. Already, new treatments targeting genetic alterations in other, less common driver oncogenes are being evaluated in lung cancer, and testing for these may be addressed in future versions of these guidelines.

Potential Therapies for Anaplastic Lymphoma Kinase-Driven Tumors in Children: Progress to Date

Anaplastic lymphoma kinase (ALK) is an oncogenic tyrosine kinase that is deregulated due to a variety of molecular mechanisms in pediatric cancer. They include chromosomal translocations, activation mutations, and gene amplifications. Since the initial discovery of ALK as an oncogenic tyrosine kinase involved in the chromosomal translocation t(2, 5)(p23;q35) in 1994, more than 20 translocation partners of ALK have been identified in various cancers. Furthermore, deregulation of ALK tyrosine kinase activity is critical for the pathogenesis of several other pediatric tumors, including neuroblastomas and inflammatory myofibroblastic tumors. The recent discovery of ALK translocations in adult lung cancer patients (non-small cell lung cancer) has accelerated the development of inhibitors of ALK tyrosine kinase as therapeutic agents. While excellent clinical response has been observed in many patients, the acquisition of clinical resistance to ALK inhibition highlights the need for development of second-generation ALK kinase inhibitors and/or combination therapies that target downstream signaling mediators or antibody drug conjugates. This article provides an update on the spectrum of ALK-driven tumors in the pediatric population and the potential therapies which target these tumors.

A retrospective analysis of the prevalence of EGFR or KRAS mutations in patients (pts) with crizotinib-naïve and crizotinib-resistant, ALK-positive non-small cell lung cancer (NSCLC).

8083 Background: Anaplastic lymphoma kinase (ALK) gene rearrangements define a distinct molecular subset of NSCLC. Recently, several studies have reported that ALK+ pts occasionally harbor concomitant mutations in other oncogenic drivers. Methods: We retrospectively analyzed tumor genotyping data from 1,683 pts with NSCLC seen at 3 U.S. centers from 2009 – 2012 to determine rates of overlapping alterations in EGFR, KRAS and ALK. Mutations in EGFR and KRAS were mainly identified using the SNaPshot multiplexed assay (>95% of cases). ALK FISH was performed in all cases. To determine if this prevalence is impacted by crizotinib, we also updated our earlier analysis (Katayama et al., Sci Transl Med, 2012) of a series of repeat biopsy specimens from 34 crizotinib-resistant, ALK+ pts. Resistant specimens were examined using ALK FISH, SNaPshot, and direct sequencing of the ALK tyrosine kinase domain (TKD). Results: Screening identified 301 (17.8%) EGFR mutations, 465 (27.6%) KRAS mutations, and 75 (4.4%) ALK rearrangements. EGFR mutations and ALK rearrangements were mutually exclusive. 4 pts with KRAS mutations also had abnormal ALK FISH patterns, involving isolated 5’ green probes (3/4 cases) and an isolated 3’ red probe that was unusually small (1/4 cases). Sufficient tissue was available for confirmatory ALK immunohistochemistry (clone 5A4, Novacastra, UK) in 3 of these cases, all of which were negative for ALK expression. Among pts with ALK+ NSCLC and acquired crizotinib resistance, repeat biopsy specimens remained ALK fusion positive in 28/28 (100%) cases. Secondary mutations in the ALK TKD (1151Tins, L1196M, G1202R, S1206Y, and G1269A) were identified in 7/34 (20.6%) cases. L1196M was the most common secondary mutation (3/34, 8.8% cases). ALK gene amplification was present in 3/28 (10.71%) pts. No EGFR or KRAS mutations were identified in 23 crizotinib-resistant, ALK+ pts with sufficient tissue for testing. Conclusions: Functional ALK rearrangements were mutually exclusive with EGFR and KRAS mutations in a large Western patient population. This lack of overlap was also observed in ALK+ pts with acquired resistance to crizotinib.

Anaplastic lymphoma kinase gene rearrangement and non-small cell lung cancer management: a step forward in personalized therapy

Dear Editor, We greatly enjoyed reading the thought-provoking article by Lopes et al. in a recent issue of your esteemed journal (1). Interestingly, in recent years, data have emerged regarding a role for EML4 (echinoderm microtubule-associated protein-like 4)–ALK (anaplastic lymphoma kinase) in personalized treatment for advanced non-small-cell lung cancer (NSCLC) (2,3). EML4-ALK undergoes constitutive dimerization through the interaction between the coil-coil domains within the EML4 region of each monomer, thereby activating ALK and promoting oncogenic activity (4). EML4-ALK fusion, EGFR, and KRAS mutations are all mutually exclusive, implicating ALK rearrangement as a potential therapeutic target in EGFR wild-type and KRAS wild-type lung cancers (5). Nevertheless, Lopes' study (1) raises some interesting questions. Was patient selection for the study carried out appropriately? Did the study provide a representative sample of the Latin-American population, as described in the paper? Could immunohistochemistry be substituted for fluorescence in situ hybridization? Which technique is more appropriate for clinical practice? Should we perform this analysis in a specific subset of patients or in all advanced NSCLC patients? In addition, it is interesting to speculate whether EML4-ALK-targeted therapies may be relevant for clinicians, although patients positive for the EML4–ALK fusion protein frequently present at an advanced clinical stage, and their tumors demonstrate a solid adenocarcinoma pattern and signet ring cells (5). Furthermore, the presence of the EML4-ALK fusion oncoprotein is associated with nonsmokers or light smokers and is more frequent in younger patients (5). Thus, young, non-smoking patients and patients with adenocarcinoma tumor histology may indeed benefit from an EML4-ALK diagnostic test. Recently, the U.S. Food and Drug Administration approved crizotinib, which is a small-molecule inhibitor of the ALK tyrosine kinase, as a treatment for patients with locally advanced or metastatic NSCLC expressing the EML4–ALK fusion protein (2). The results were recently reported for a clinical trial in which 82 ALK-positive patients were evaluated for the therapeutic efficacy of crizotinib (6). The results were quite promising, demonstrating an overall response rate of 57% (46 partial responses and 1 complete response) and a 33% stable disease rate (27 patients) (2). Of the 82 patients, 63 (77%) continued to receive crizotinib at the time of data cut off, and the estimated probability of a 6-month progression-free survival was 72% (2). Thus, the refined understanding of the NSCLC molecular profile described over the last decade has proven to be an important tool to help medical oncologists develop new approaches for NSCLC treatment. Despite the small frequency of advanced NSCLC patients who present with EML4-ALK fusion (2.4 to 6.7%) (1), we believe that all patients in this setting should have the opportunity to receive such innovative therapies and approaches. However, further studies are warranted to establish the appropriate pharmaco-economic profiles.

Abstract 4444: Switching oncogene signaling in a highly selective ALK tyrosine kinase inhibitor CH5424802 resistant cells.

Abstract We experienced dramatic responses to crizotinib, a multitargeted tyrosine kinase inhibitor including anaplastic lymphoma kinase (ALK), MET, and ROS1 in patients with non-small cell lung cancer (NSCLC) harboring ALK fusion gene; however, majority of the patients relapsed until around 10 months. The emergence of resistance to crizotinib is reportedly conferred by genetic alterations, such as ALK kinase domain mutation, ALK copy number gain or activation of the bypass pathways. CH5424802 (Chugai Pharmaceutical Co., Ltd.) is a new, highly selective ALK tyrosine kinase inhibitor that partially overcome acquired resistance to crizotinib, thereby inhibiting crizotinib resistant mutations of ALK in a preclinical experiment (Cancer Cell 19, 2011: 679-90) A phase II study of CH5424802 demonstrated 93.5% response rate (JLCS 2012); however, we also encountered acquired resistance phenomenon. To elucidate the acquired resistant mechanisms, we established CH5424802-resistant H2228 cells harboring EML4-ALK fusion gene under the step-wise continuous exposures of CH5424802. Six sublines that could grow in the presence of 1 μmol/L of CH5424802 (designated H2228CHR clones -1, -2, -3, -5, -6, and -9) were obtained. All sublines revealed more than 30-fold resistance to CH5424802 (50% inhibitory concentration [IC50]: 1.0-4.8 μmol/L) compared with the parental H2228 cells (IC50: 0.03 μmol/L). In addition, the sublines similarly indicated resistance to Crizotinib. H2228CHR clones showed the constitutive phosphorylation of Akt and Erk1/2 even in the presence of CH5424802, whereas phosphorylation of Akt and Erk1/2 was markedly decreased in the parental H2228 cells. Surprisingly, ALK fusion gene disappeared in all H2228 CHR clones, which were confirmed immunostaining, reverse transcription PCR, FISH and Western blotting. H2228 CHR clones -1 and -2 presented higher phosphorylated levels of epidermal growth factor receptor (EGFR) than the parental H2228 cells and showed sensitivity to EGFR tyrosine kinase inhibitors, gefitinib and erlotinib. Furthermore, we also established CH5424802-resistant ABC-11CHR cells derived from ABC-11 cells from pleural effusion of an ALK inhibitor naïve patient. In ABC-11CHR cells, phosphorylated ALK was markedly decreased and phosphorylated MET was increased. In conclusion, signal switching from ALK to other tyrosine kinase receptors induced resistance to a highly selective ALK tyrosine kinase inhibitor CH5424802 in NSCLC cell lines harboring ALK fusion gene. Citation Format: Hideko Isozaki, Eiki Ichihara, Masayuki Yasugi, Takashi Ninomiya, Yoshihiro Honda, Toshi Murakami, Daisuke Minami, Yuka Kato, Kenitiro Kudo, Akiko Sato, Katsuyuki Hotta, Nagio Takigawa, Toshiaki Sendo, Mitsune Tanimoto, Katsuyuki Kiura. Switching oncogene signaling in a highly selective ALK tyrosine kinase inhibitor CH5424802 resistant cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4444. doi:10.1158/1538-7445.AM2013-4444

Molecular Testing Guideline for Selection of Lung Cancer Patients for EGFR and ALK Tyrosine Kinase Inhibitors: Guideline from the College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology

To establish evidence-based recommendations for the molecular analysis of lung cancers that are required to guide EGFR- and ALK-directed therapies, addressing which patients and samples should be tested, and when and how testing should be performed. Three cochairs without conflicts of interest were selected, one from each of the 3 sponsoring professional societies: College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology. Writing and advisory panels were constituted from additional experts from these societies. Three unbiased literature searches of electronic databases were performed to capture published articles from January 2004 through February 2012, yielding 1533 articles whose abstracts were screened to identify 521 pertinent articles that were then reviewed in detail for their relevance to the recommendations. EVIDENCE was formally graded for each recommendation. Initial recommendations were formulated by the cochairs and panel members at a public meeting. Each guideline section was assigned to at least 2 panelists. Drafts were circulated to the writing panel (version 1), advisory panel (version 2), and the public (version 3) before submission (version 4). The 37 guideline items address 14 subjects, including 15 recommendations (evidence grade A/B). The major recommendations are to use testing for EGFR mutations and ALK fusions to guide patient selection for therapy with an epidermal growth factor receptor (EGFR) or anaplastic lymphoma kinase (ALK) inhibitor, respectively, in all patients with advanced-stage adenocarcinoma, regardless of sex, race, smoking history, or other clinical risk factors, and to prioritize EGFR and ALK testing over other molecular predictive tests. As scientific discoveries and clinical practice outpace the completion of randomized clinical trials, evidence-based guidelines developed by expert practitioners are vital for communicating emerging clinical standards. Already, new treatments targeting genetic alterations in other, less common driver oncogenes are being evaluated in lung cancer, and testing for these may be addressed in future versions of these guidelines.

Cell culture andDrosophilamodel systems define three classes of anaplastic lymphoma kinase mutations in neuroblastoma

SUMMARYNeuroblastoma is a childhood extracranial solid tumour that is associated with a number of genetic changes. Included in these genetic alterations are mutations in the kinase domain of the anaplastic lymphoma kinase (ALK) receptor tyrosine kinase (RTK), which have been found in both somatic and familial neuroblastoma. In order to treat patients accordingly requires characterisation of these mutations in terms of their response to ALK tyrosine kinase inhibitors (TKIs). Here, we report the identification and characterisation of two novel neuroblastoma ALK mutations (A1099T and R1464STOP), which we have investigated together with several previously reported but uncharacterised ALK mutations (T1087I, D1091N, T1151M, M1166R, F1174I and A1234T). In order to understand the potential role of these ALK mutations in neuroblastoma progression, we have employed cell culture-based systems together with the model organism Drosophila as a readout for ligand-independent activity. Mutation of ALK at position 1174 (F1174I) generates a gain-of-function receptor capable of activating intracellular targets such as ERK (extracellular signal regulated kinase) and STAT3 (signal transducer and activator of transcription 3) in a ligand-independent manner. Analysis of these previously uncharacterised ALK mutants and comparison with ALKF1174 mutants suggests that ALK mutations observed in neuroblastoma fall into three classes. These classes are: (i) gain-of-function ligand-independent mutations such as ALKF1174l, (ii) kinase-dead ALK mutants, e.g. ALKI1250T (Schönherr et al., 2011a) and (iii) ALK mutations that are ligand-dependent in nature. Irrespective of the nature of the observed ALK mutants, in every case the activity of the mutant ALK receptors could be abrogated by the ALK inhibitor crizotinib (Xalkori/PF-02341066), albeit with differing levels of sensitivity.

Current Status of Targeted Therapy for Anaplastic Lymphoma Kinase–Rearranged Non–Small Cell Lung Cancer

The identification of chromosomal rearrangements involving the anaplastic lymphoma kinase (ALK) gene in ~3-5% of non-small cell lung cancer (NSCLC) tissues and the demonstration that the first-in-class ALK tyrosine kinase inhibitor, crizotinib, can effectively target these tumors represent a significant advance in the evolution of personalized medicine for NSCLC. Single-arm studies demonstrating rapid and durable responses in the majority of ALK-positive NSCLC patients treated with crizotinib have been followed by a randomized phase III clinical trial in which superiority of crizotinib over chemotherapy was seen in previously treated ALK-positive NSCLC patients. However, despite the initial responses, most patients develop acquired resistance to crizotinib. Several novel therapeutic approaches targeting ALK-positive NSCLC are currently under evaluation in clinical trials, including second-generation ALK inhibitors, such as LDK378, CH5424802 (RO5424802802), and AP26113, and heat shock protein 90 inhibitors.

Oncology in 2012: from personalized medicine to precision medicine

Although oncologists seem unable to name the new paradigm of cancer treatment, a certain trend seems to take shape in favor of the precision medicine. To paraphrase Mickael P. Link, President of the American Society of Clinical Oncology, precision medicine identifies and makes the most of the weaknesses of cancer genetics so that the tumoral growth can be stopped. Doubtless, major advances in this oncologic medicine of precision have been observed throughout 2012. These advances have first of all affected the theranostics. This is how two new oncogenic drivers were discovered in lung adenocarcinoma. The first one ROS1 is situated in 6q. 22 chromosome. ROS1 is observed in 1 % of the cases and would seem to be sensitive to the anaplastic lymphoma kinase (ALK) and C-MET inhibitors [1]. Kinase inhibitor fusion 5B—rearranged during transfection [1], the second, is observed in 1.3 % of non-small cell lung cancer (NSCLC). Two aspects of these discoveries are important to underline because there are agents targeting these molecular modifications (crizotinib and a tyrosine kinase inhibitor anti-RET) and also because these molecular targets exist in other tumoral types: glioblastoma or cholangiocarcinoma for ROS1. In addition to this progress in theranostics, numerous advances have concerned new, soon-to-be-available agents in oncology practice. This is the way crizonitinb, a powerful inhibitor of ALK tyrosine kinase receptor [2], demonstrates its efficacy in the ALK-positive NSCLC previously treated with first-line platinum-based chemotherapy. This treatment is superior to standard second-line chemotherapy with a striking hazard ratio (HR) of 0.49 [3]. Regorafenib [4] offers a new therapeutic option in metastatic colorectal cancers in second-line treatment and in GIST. Cabozantinib, a new potent inhibitor of VEGFR and MET and VEGFr2 [5], shows promise in several tumor types, such as prostate, breast, and hepatocarcinoma. Dabrafenib, a new anti-RAF agent can achieve an HR of 0.35 for progression-free survival in patients with metastatic melanoma [6]. MEK blockade is one of the last novelties in signaling. Take the example of trametinib, which compares favorably to chemotherapy in metastatic melanoma (HR0 0.54 (0.32–0.92)) [7]. Signaling pathways are not the only process that is “targetable.” Immunotherapeutics is currently regaining its former glory. The stimulation of the immune system against tumors through targeting regulatory T cells is taking shape. It is now evident that monoclonal antibodies directed against cytotoxic T lymphocyte antigen 4 or programmed cell death 1 represent dramatic progress in the management of melanoma [8]. Thanks to new agents, there is even more hope for our patients:

SY14-3 - Treatment of EML4-ALK Rearranged NSCLC

ABSTRACT Rearrangements in the anaplastic lymphoma kinase (ALK) gene have been detected in ∼3–7% of patients with non-small-cell lung cancer (NSCLC). Crizotinib, an ALK tyrosine kinase inhibitor (TKI), is clinically effective in NSCLC patients with ALK rearrangements. In the phase I clinical trial, a radiographic response rate of 54% was observed. Crizotinib was recently approved by the FDA for the treatment of ALK-positive NSLC. Current clinical trials of crizotinib include two randomized phase III clinical trials. The first compares crizotinib to second-line chemotherapy (either pemetrexed or docetaxel) in ALK-positive NSCLC patients that have failed first-line platinum-based chemotherapy. The second trial compares crizotinib with cisplatin (or carboplatin) and pemetrexed in treatment naive advanced NSCLC ALK positive patients. In addition to ALK TKIs, inhibitors of heat shock protein (HSP) 90 have demonstrated some clinical activity in EML4-ALK NSCL and are undergoing additional clinical evaluation. However, despite the impressive clinical efficacy of crizotinib, it is fully anticipated that acquired drug resistance will emerge. Drug resistance mechanisms can in general be divided into two categories: those that result in secondary mutations in the target itself (i.e. ALK) or those that activate parallel signaling pathways. For ALK-positive cancers both mechanisms have been identified to date. Secondary mutations in ALK to date include L1196M, F1174L, C1156Y and L1152R. One example of an alternative signaling pathway that leads to ALK TKI resistance is EGFR. In vitro, some ALK TKI-resistant cell lines have activated EGFR signaling (via EGFR ligands) that promote crizotinib resistance. The knowledge of a specific drug resistance mechanism is important for choosing the next treatment of ALK positive patients that have developed Crizotinib resistance. In those with evidence of an ALK secondary mutation a more potent ALK inhibitor may be effective. There are several such in preclinical and at least three in phase I clinical development. In cancers harboring activated signaling pathways (such as EGFR) an alternative strategy, a combination of an ALK and EGFR inhibitor, may be effective. A phase I clinical trial is underway combining Crizotinib with the pan-ERBB inhibitor PF299804.