Echinoderm Microtubule-associated Protein Research Articles

Establishment of potential reference measurement procedure and reference materials for EML4-ALK fusion variants measurement

Echinoderm microtubule-associated protein 4 (EML4) - anaplastic lymphoma kinase (ALK) fusion gene detection is of great significance in personalized tumor treatment. With the development of EML4-ALK fusion variants detection, it is necessary to establish traceability to ensure the consistency and comparability of its detection results in clinical practice. The establishment of traceability relies on SI traceable reference materials (RMs) and potential reference measurement procedures (RMPs). In this study, a potential RMP for the quantitative detection of V1 and V3 fusion mutations and the reference type (ALK-ref, including wild type, V1 and V3 mutant type) based on reverse transcription dPCR (RT-dPCR) and EML4-ALK fusion gene RMs were established. The proposed potential RMP has high specificity, good inter-laboratory reproducibility (CV < 7.3%) and good linear relationship (0.92 < slope < 1.06, R2 ≧ 0.99). The limit of detection (LoD) of V1, V3, and ALK-ref are 2 copies/reaction, 2 copies/reaction, and 1 copy/reaction, respectively. Interlaboratory studies using the EML4-ALK RMs and potential RMP showed that participating laboratories can produce consistent copy concentrations of fusion variant and ALK-ref as well as the ratio of EML4-ALK/ALK-ref. The established potential RMP with high specificity and accuracy can be used to characterize the EML4-ALK RMs, and the potential RMP and RM are useful to establish the traceability of EML4-ALK fusion measurement to improve the comparability and consistency in clinical tests.

Forebrain Eml1 depletion reveals early centrosomal dysfunction causing subcortical heterotopia.

Subcortical heterotopia is a cortical malformation associated with epilepsy, intellectual disability, and an excessive number of cortical neurons in the white matter. Echinoderm microtubule-associated protein like 1 (EML1) mutations lead to subcortical heterotopia, associated with abnormal radial glia positioning in the cortical wall, prior to malformation onset. This perturbed distribution of proliferative cells is likely to be a critical event for heterotopia formation; however, the underlying mechanisms remain unexplained. This study aimed to decipher the early cellular alterations leading to abnormal radial glia. In a forebrain conditional Eml1 mutant model and human patient cells, primary cilia and centrosomes are altered. Microtubule dynamics and cell cycle kinetics are also abnormal in mouse mutant radial glia. By rescuing microtubule formation in Eml1 mutant embryonic brains, abnormal radial glia delamination and heterotopia volume were significantly reduced. Thus, our new model of subcortical heterotopia reveals the causal link between Eml1's function in microtubule regulation and cell position, both critical for correct cortical development.

A CRISPR activation screen identifies FBXO22 supporting targeted protein degradation.

Targeted protein degradation (TPD) represents a potent chemical biology paradigm that leverages the cellular degradation machinery to pharmacologically eliminate specific proteins of interest. Although multiple E3 ligases have been discovered to facilitate TPD, there exists a compelling requirement to diversify the pool of E3 ligases available for such applications. Here we describe a clustered regularly interspaced short palindromic repeats (CRISPR)-based transcriptional activation screen focused on human E3 ligases, with the goal of identifying E3 ligases that can facilitate heterobifunctional compound-mediated target degradation. Through this approach, we identified a candidate proteolysis-targeting chimera (PROTAC), 22-SLF, that induces the degradation of FK506-binding protein 12 when the transcription of FBXO22 gene is activated. Subsequent mechanistic investigations revealed that 22-SLF interacts with C227 and/or C228 in F-box protein 22 (FBXO22) to achieve target degradation. Lastly, we demonstrated the versatility of FBXO22-based PROTACs by effectively degrading additional endogenous proteins, including bromodomain-containing protein 4 and the echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase fusion protein.

ALK fusion NSCLC oncogenes promote survival and inhibit NK cell responses via SERPINB4 expression

Anaplastic lymphoma kinase (ALK) fusion variants in Non-Small Cell Lung Cancer (NSCLC) consist of numerous dimerizing fusion partners. Retrospective investigations suggest that treatment benefit in response to ALK tyrosine kinase inhibitors (TKIs) differs dependent on the fusion variant present in the patient tumor. Therefore, understanding the oncogenic signaling networks driven by different ALK fusion variants is important. To do this, we developed controlled inducible cell models expressing either Echinoderm Microtubule Associated Protein Like 4 (EML4)-ALK-V1, EML4-ALK-V3, Kinesin Family Member 5B (KIF5B)-ALK, or TRK-fused gene (TFG)-ALK and investigated their transcriptomic and proteomic responses to ALK activity modulation together with patient-derived ALK-positive NSCLC cell lines. This allowed identification of both common and isoform-specific responses downstream of these four ALK fusions. An inflammatory signature that included upregulation of the Serpin B4 serine protease inhibitor was observed in both ALK fusion inducible and patient-derived cells. We show that Signal transducer and activator of transcription 3 (STAT3), Nuclear Factor Kappa B (NF-κB) and Activator protein 1 (AP1) are major transcriptional regulators of SERPINB4 downstream of ALK fusions. Upregulation of SERPINB4 promotes survival and inhibits natural killer cell-mediated cytotoxicity, which has potential for therapeutic impact targeting the immune response together with ALK TKIs in NSCLC.

Expression levels of NPPB, ITGB6, CPNE4, EML5, and ITSN1 in fresh exudates swabbed from critically colonised and infected full-thickness wounds in rats.

Pressure injury management requires reliable identification of critical colonisation due to lack of infection signs. Our research group previously proposed the mRNAs natriuretic peptide B (Nppb), integrin subunit beta 6 (Itgb6), copine 4 (Cpne4), echinoderm microtubule-associated protein like 5, and intersectin 1 as candidate markers in pooled exudates of critically colonised wounds. However, it is unclear whether mRNAs or proteins of the candidate genes would be suitable as biomarkers in fresh exudate. Therefore, this study aimed to evaluate the validity of the mRNAs and proteins as fresh exudate markers for critical colonisation. Three wound models of normal healing, critical colonisation, and infection were created in rats. Fresh swab-collected exudates were collected, and mRNA and protein expression levels were measured. In the fresh wound exudates, the detection frequency of Itgb6 tended to decrease in the critically colonised and infected wounds (P=.067), and those of Cpne4 and Nppb tended to be lower in the infected wounds than in the normal healing and critically colonised wounds (P=.006 and .067, respectively). In contrast, there was no difference in protein expression in the exudates. This study suggests that Itgb6 mRNA in fresh exudates is a promising biomarker for critical colonisation.

Abstract B032: Deciphering the mechanisms of environmentally mediated Alectinib resistance in ALK+NSCLC

Abstract Non-small cell lung cancer (NSCLC) is a devastating disease with a subset containing targetable oncogenic mutations that strongly benefit from targeted therapies. 3-7% of NSCLC patients bear an amplification of the Anaplastic lymphoma kinase (ALK) and Echinoderm microtubule-associated protein like 4 (EML4) fusion gene. These tumors typically show strong durable responses to first line therapy, Alectinib, an ALK tyrosine kinase inhibitors (ALK-TKIs). However, there is a high degree of patient-to-patient variability in the level of response to this drug; with some patients being innately resistant or less sensitive. Moreover, weak initial sensitivity to therapy has been linked to poor long-term responses with early acquisition of resistance. Thus, to improve clinical outcomes, we need to understand the mechanisms that lead to this reduced sensitivity to ALK inhibition. While, historically, reduced sensitivity to ALK TKI was thought to reflect cell intrinsic mechanisms, a growing body of evidence points to the importance of tumor microenvironmentally (TME) mediated mechanisms. We fortuitously encountered an observation of strong environment-mediated desensitization to Alectinib. We found that combining Alectinib with a small molecule inhibitor, Jumonji histone demethylase inhibitor, JIB04 in vitro overcomes tolerance, preventing ALK+NSCLC cells from developing resistance to Alectinib. Surprisingly, our validation experiments in mouse models revealed the opposite effect, where, JIB04 desensitized tumors to ALK inhibition. The disparity in the in vitro versus the in vivo response indicates the contribution of the TME in the reduced Alectinib sensitivity observed in vivo. The resistance phenotype in Alectinib+JIB04 treated mice was strongly associated with extensive inflammation in the tumor proximal tissues, along with higher circulating levels of inflammatory markers, IL6, LIF and monocytes, suggesting that inflammation may play a role in mediating this resistance. This is consistent with the recent findings where the markers of systemic inflammation such as Neutrophil to Lymphocyte ratio (NLR) have been associated with poor prognosis in ALK+NSCLC. While the mechanistic understanding is not well characterized, our model may help us explore the mechanisms of this correlation. Thus, our objective is to understand the underlying microenvironmental mechanism of this resistance to Alectinib. We hypothesize that Alectinib+JIB04 associated inflammation reduces ALK+NSCLC’s sensitivity to Alectinib. We envision two possibilities, either inflammation may directly affect resistance to Alectinib or indirectly where inflammation enhances tumor cell plasticity that then leads to resistance. We posit that the underlying molecular mechanisms of Alectinib+JIB04 mediated resistance are likely to be relevant to a subset of patients with poor initial responses with weak sensitivity or innate resistance to Alectinib observed in the clinics and will improve our knowledge about microenvironmental regulation of resistance. Citation Format: Pragya Kumar, Virginia Turati, Andriy Marusyk. Deciphering the mechanisms of environmentally mediated Alectinib resistance in ALK+NSCLC [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr B032.

F-circEA1 regulates cell proliferation and apoptosis through ALK downstream signaling pathway in non-small cell lung cancer

Studies have confirmed that circular RNA (circRNA) has a stable closed structure, which plays an important role in the progression of tumors. Cancers with positive fusion genes can produce associated fusion circRNA (F-cirRNA). However, there are no reports concerning a role for F-circRNA of the echinoderm microtubule associated-protein like 4-anaplastic lymphoma kinase variant 1 (EML4-ALK1) in non-small cell lung cancer (NSCLC). Our study confirmed the existence of fusion circEA1 (F-circEA1) in NCI-H3122 cells (carrying the EML4-ALK1 gene), F-circEA1 was expressed both in the cytoplasm and nucleus as determined by fluorescence in situ hybridization (FISH) and Sanger sequencing. CCK8 and transwell assays showed that F-circEA1 was beneficial to cell proliferation, metastasis, and invasion. Overexpression of F-circEA1 can also promote cell proliferation, migration and invasion in A549 and SPCA1 cells (non-small cell lung cancer cell line not carrying the EML4-ALK1 gene). Interference with F-circEA1, induced cell cycle arrest and promoted apoptosis as determined by flow cytometry, and increased drug sensitivity to crizotinib in H3122 cells. F-circEA1 directly affected the expression of parental gene EML4-ALK1. Further research found that F-circEA1 can affect the downstream signaling pathway of ALK. In vivo, the growth rate of xenogeneic tumors was reduced and the protein expression level of EML4-ALK1 was significantly decreased in transplanted tumors measured by immunohistochemistry (IHC) after interference with F-circEA1. In conclusion, F-circEA1 can be considered as a proto-oncogene that regulates cell proliferation and apoptosis by affecting the expression of the parental gene EML4-ALK1 and its ALK downstream signaling pathway in non-small cell lung cancer.

Echinoderm Microtubule Associated Protein Like 1 Is Indispensable for Oocyte Spindle Assembly and Meiotic Progression in Mice.

Completion of the first meiosis is an essential prerequisite for producing a functionally normal egg for fertilization and embryogenesis, but the precise mechanisms governing oocyte meiotic progression remains largely unclear. Here, we report that echinoderm microtubule associated protein (EMAP) like 1 (EML1), a member of the conserved EMAP family proteins, plays a crucial role in the control of oocyte meiotic progression in the mouse. Female mice carrying an ENU-induced nonsense mutation (c.1956T > A; p.Tyr652∗) of Eml1 are infertile, and the majority of their ovulated oocytes contain abnormal spindles and misaligned chromosomes. In accordance with the mutant oocyte phenotype, we find that EML1 is colocalized with spindle microtubules during the process of normal oocyte meiotic maturation, and knockdown (KD) of EML1 by specific morpholinos in the fully grown oocytes (FGOs) disrupts the integrity of spindles, and delays meiotic progression. Moreover, EML1-KD oocytes fail to progress to metaphase II (MII) stage after extrusion of the first polar body, but enter into interphase and form a pronucleus containing decondensed chromatins. Further analysis shows that EML1-KD impairs the recruitment of γ-tubulin and pericentrin to the spindle poles, as well as the attachment of kinetochores to microtubules and the proper inactivation of spindle assembly checkpoint at metaphase I (MI). The loss of EML1 also compromises the activation of maturation promoting factor around the time of oocyte resumption and completion of the first meiosis, which, when corrected by WEE1/2 inhibitor PD166285, efficiently rescues the phenotype of oocyte delay of meiotic resumption and inability of reaching MII. Through IP- mass spectrometry analysis, we identified that EML1 interacts with nuclear distribution gene C (NUDC), a critical mitotic regulator in somatic cells, and EML1-KD disrupts the specific localization of NUDC at oocyte spindles. Taken together, these data suggest that EML1 regulates acentrosomal spindle formation and the progression of meiosis to MII in mammalian oocytes, which is likely mediated by distinct mechanisms.

Advances in Drug Resistance Mechanisms and Prognostic Markers of Targeted Therapy in ALK-positive Non-small Cell Lung Cancer

棘皮动物微管相关类蛋白4-间变性淋巴瘤激酶(echinoderm microtubule-associated protein like 4-anaplastic lymphoma kinase, EML4-ALK)融合占非小细胞肺癌(non-small cell lung cancer, NSCLC)患者的3%-5%。随着对该驱动基因的深入研究，以Crizotinib为代表的ALK抑制剂逐渐被开发并应用于临床。然而，不同患者对ALK靶向治疗的反应存在差异，且多数ALK靶向治疗患者最终会不可避免地出现耐药，导致肿瘤进展。利用预后标志物监测患者疗效及时改变治疗方案，以及根据耐药机制选择个体化的后续治疗，可以有效地改善患者的预后。本文将对ALK抑制剂的耐药机制以及相关的预后标志物展开综述，探讨ALK靶向治疗疗效预测以及耐药患者后续治疗方案的选择。

Abstract 4900: An ALK fusion gene regulates different signaling pathways in mortal versus immortalized normal human cells for cellular senescence and transformation

Abstract Accumulating results of clinical trials lead targeted therapies to be the first choice for unresectable or recurrent lung cancer with driver mutations. Echinoderm Microtubule Associated Protein Like 4 (EML4) - Anaplastic lymphoma kinase (ALK) fusion is known as such a driver mutation. It presents in 3-6% of non-small cell lung carcinoma (NSCLC). EML4-ALK fusion protein generate the constitutive ALK kinase activity in NSCLC. The basic understanding of EML4-ALK remains insufficient due to the lack of functional studies using normal human cells. We investigated the role of EML4-ALK in mortal and immortalized normal human cells. The expression of EML4-ALK in normal, mortal human fibroblasts caused accumulated DNA damage, telomere shortening and the early induction of cellular senescence with senescence-associated beta-galactosidase activity and upregulation of p16INK4A and p21WAF1. In contrast, when EML4-ALK was expressed in telomerase reverse transcriptase (hTERT)-immortalized normal human fibroblasts and bronchial epithelial cells, the cells showed accelerated proliferation in vitro and anchorage-independent growth in soft agar, revealing its transformation activity. No chromosome aberrations, no mutations or loss of p53, nor impairment of the p16INK4A response was associated with this in vitro transformation, likely reflecting certain clinical features of EML4-ALK-positive NSCLC. In both mortal and immortalized cells, EML4-ALK induced the phosphorylation of STAT3, which is involved in both cellular senescence and transformation. Our data validate that EML4-ALK functions as an oncogene, although an additional oncogenic event(s) seems to be required for full tumorigenicity in vivo. This study also suggests that telomerase-mediated immortalization manifests the oncogenic activity of EML4-ALK, switching from its senescence-inducing activity. The RNA-seq analysis showed that the STAT3-induced cytokine/interferon signaling pathways were most significantly upregulated by EML4-ALK in mortal fibroblasts, consistent with accumulated DNA damage and senescence induction. The blood coagulation pathway activated by EML4-ALK in hTERT-immortalized cells may contribute to increased risk of disseminated intravenous coagulation in patients with EML4-ALK-positive cancer. These results suggest that EML4-ALK regulates the different signaling pathways in mortal versus immortalized normal human cells to induce the different cellular outcomes. Citation Format: Masaru Matsumoto, Akihiko Miyanaga, Jessica Beck, Izumi Horikawa, Mohammed Khan, Delphine Lissa, Masahiro Seike, Akihiko Gemma, Hiroyuki Mano, Curtis Harris. An ALK fusion gene regulates different signaling pathways in mortal versus immortalized normal human cells for cellular senescence and transformation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4900.

Participation of EML6 in the regulation of oocyte meiotic progression in mice.

The generation of a high-quality egg for reproduction requires faithful segregation of chromosome during oocyte meiosis. Here, we report that echinoderm microtubule-associated protein like 6 (EML6) is highly expressed in oocytes, and responsible for accurate segregation of homologous chromosomes in mice. Quantitative real-time RT-PCR and immunohistochemistry analyses revealed that EML6 was predominantly expressed by oocytes in the ovaries. Whole mount immunofluorescent staining showed that EML6 was colocalized with spindle microtubules in oocytes at various stages after meiotic resumption. This specialized localization was disrupted by nocodazole, the microtubule destabilizer, while enhanced by Taxol, a microtubule stabilizing reagent. In vivo knockdown of Eml6 expression by the specific siRNA resulted in chromosome misalignment and alteration in spindle dimension at both metaphase Ⅰ and Ⅱ stages, as well as the increased aneuploidy in the mature oocytes. Thus, these data suggest that EML family proteins participate in the control of oocyte meiotic division.

Abstract 4648: Cellular senescence and transformation induced by an oncogenic EML4-ALK fusion gene in normal and immortalized human cells

Abstract Background: Chromosomal inversions involving Anaplastic lymphoma kinase (ALK) and Echinoderm Microtubule Associated Protein Like 4 (EML4) generate a fusion protein EML4-ALK with the constitutive ALK kinase activity in non-small cell lung carcinoma (NSCLC). The basic understanding of EML4-ALK remains insufficient due to the lack of functional studies using normal human cells. Material and Method: We investigate the activities of EML4-ALK in mortal and immortalized normal human cells. Results: The expression of EML4-ALK in normal, mortal human fibroblasts caused, through its ALK kinase activity, the early induction of cellular senescence with senescence-associated β-galactosidase activity, upregulation of p16INK4A and p21WAF1, telomere shortening and fusions, and accumulated DNA damage. In contrast, when EML4-ALK was expressed in telomerase reverse transcriptase (hTERT)-immortalized normal human fibroblasts, the cells showed accelerated proliferation and anchorage-independent growth, revealing its transformation activity. No chromosome aberration, no mutation or loss of p53, nor impairment of the p16INK4A response was associated with this transformation, likely reflecting clinical features of EML4-ALK-positive NSCLC. In both mortal and immortalized cells, EML4-ALK induced the phosphorylation of STAT3, which is involved in both cellular senescence and transformation. EML4-ALK was also able to transform hTERT-immortalized human bronchial epithelial cells, a cell type relevant to NSCLC. Conclusions: Our data not only validate that EML4-ALK functions as an oncogene in human cells, but also suggest that telomerase-mediated immortalization manifests the oncogenic activity of EML4-ALK, switching from its senescence-inducing activity. This study also provides the isogenic pairs of human cell lines with and without EML4-ALK as an in vitro model for screening and testing candidate drugs. Citation Format: Akihiko Miyanaga, Izumi Horikawa, Masaru Matsumoto, Takahiro Oike, Jessica Beck, Hiromi Tanaka, Ana I. Robles, Masahiro Seike, Akihiko Gemma, Curtis C. Harris. Cellular senescence and transformation induced by an oncogenic EML4-ALK fusion gene in normal and immortalized human cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4648.

Abstract 2982: Monomerization of ALK fusion proteins as a therapeutic strategy in ALK rearranged cancers

Abstract Introduction: Since the discovery of the anaplastic lymphoma kinase (ALK) and echinoderm microtubule-associated protein like-4 (EML4) fusion protein, ALK tyrosine kinase inhibitors have been shown to have great anti-tumor activities against ALK rearranged non-small cell lung cancers (NSCLC). As previously reported, the EML4-ALK fusion proteins induce constitutive phosphorylation of ALK via the trimeric coiled-coil domain within EML4. ALK F1174L mutations known in neuroblastoma, can induce autophosphorylation without dimerization. We hypothesize that monomerization of ALK fusion proteins decreased tumorigenesis via competitive dissociation of the trimeric coiled-coil domain of EML4. Methods: We established Ba/F3 cells stably expressing wild-type (wt) ALK intracellular domain or F1174L mutant (mu) with using iDimerize system (Takara Bio). Using this system, monomer or dimer of ALK fusion proteins were controlled by adding AP20187 (Takara Bio). These Ba/F3 cells were subcutaneously injected into BALB nu/nu mice followed by intraperitoneal administration of AP20187 or Mock intermittently. A chemically synthesized peptide composed of amino acid sequences of coiled-coil domain within EML4, was administered for H3122 (EML4-ALK) or A549 (KRAS) cells and the viabilities were analyzed after 24 hours. In order to demonstrate the specificity of the peptide, a synthesized peptide in which two amino acids were substituted was also used as a control. Each peptide was solubilized in DMSO and the peptide concentration was optimized. Results: We successfully established the Ba/F3 stably expressing fusion-ALKwt, fusion-ALKmu under continuous administration of AP20187. Subsequently, ALK proteins were depolymerized by withdrawal of AP20187. The number of cells expressing ALKwt were decreased while the same of ALKmu were increased. The protein expression of phosphorylation of ALK or downstream survival signaling proteins were weakened after 24hours from AP20187 withdrawal. In fusion-ALKwt cells, tumor xenograft was developed in every mice with AP20187 (n=6) administration, while tumor was not developed in Mock controls (n=6). In fusion-ALKmu cells, tumor xenograft was developed in all mice with or without AP20187 administration (n=12). In vitro assay, cell growth of H3122 was 40% decreased by adding coiled-coil peptides, while increased cell growth was observed in control peptide groups and A549 cells. Conclusion: Monomerization of ALK intracellular domain induced cell death and suppressed tumorigenesis. The coiled-coil peptide specifically suppressed the activity of EML4-ALK cell line. Monomerization of ALK fusion proteins via coiled-coil peptide is a promising therapeutic strategy for ALK rearranged NSCLC. Citation Format: Noriko Hirai, Takaaki Sasaki, Yoshinobu Ohsaki. Monomerization of ALK fusion proteins as a therapeutic strategy in ALK rearranged cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2982.

Clinicopathological and molecular epidemiological study of lung cancer patients seen at a tertiary care hospital in Northern India.

Aims:The primary objective of this study was to estimate the clinicopathological and molecular profile of lung cancer patients along with the evaluation of their clinical characteristics at a tertiary care hospital in Northern India.Subjects and Methods:A total of 421 patients with lung cancer histology who were treated at Max Super Speciality Hospitals were included in the study. The study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki and permission was obtained from the Ethics Committee before the start of the study. Clinical characteristics and molecular profiling data were collected from the patient's medical records.Results:There were 330 (78.4%) men and 91 (21.6%) women with a median age of 62 years (range: 30–93 years). Of the 421 patients, 388 (92.2%) patients had the nonsmall cell lung cancer (NSCLC) histology whereas 33 (7.8%) patients were of SCLC histology. Histology and gender had a significant association with NSCLC and SCLC (P < 0.05). Epidermal growth factor receptor (EGFR) and echinoderm microtubule-associated protein like 4-anaplastic lymphoma kinase (EML4-ALK) fusion gene testing was done in 120 and 93 patients, respectively. Of the 120 patients, 24 (20%) cases were positive for EGFR mutations whereas EML4-ALK fusion gene was present in 8 (8.6%) out of 93 patients.Conclusions:Our study confirms the importance of molecular testing in the NSCLC patient subgroup with an aim to identify the exact molecular targets that can benefit from the newer generation of targeted therapies.

Alectinib for the management of ALK-positive non-small cell lung cancer brain metastases.

Anaplastic lymphoma kinase ( ALK ) rearrangements were first identified as an oncogenic driver in non-small cell lung cancers (NSCLC) in 2007, specifically the gene re-arrangement between ALK and echinoderm microtubule-associated protein like 4 ( EML4 ) (1).

Detection of Echinoderm Microtubule Associated Protein Like 4-Anaplastic Lymphoma Kinase Fusion Genes in Non-small Cell Lung Cancer Clinical Samples by a Real-time Quantitative Reverse Transcription Polymerase Chain Reaction Method.

Objective To establish a real-time quantitative reverse transcription polymerase chain reaction assay (qRT-PCR) for the rapid, sensitive, and specific detection of echinoderm microtubule associated protein like 4-anaplastic lymphoma kinase (EML4-ALK) fusion genes in non-small cell lung cancer. Methods The specific primers for the four variants of EML4-ALK fusion genes (V1, V2, V3a, and V3b) and Taqman fluorescence probes for the detection of the target sequences were carefully designed by the Primer Premier 5.0 software. Then, using pseudovirus containing EML4-ALK fusion genes variants (V1, V2, V3a, and V3b) as the study objects, we further analyzed the lower limit, sensitivity, and specificity of this method. Finally, 50 clinical samples, including 3 ALK-fluorescence in situ hybridization (FISH) positive specimens, were collected and used to detect EML4-ALK fusion genes using this method. Results The lower limit of this method for the detection of EML4-ALK fusion genes was 10 copies/μl if no interference of background RNA existed. Regarding the method's sensitivity, the detection resolution was as high as 1% and 0.5% in the background of 500 and 5000 copies/μl wild-type ALK gene, respectively. Regarding the method's specificity, no non-specific amplification was found when it was used to detect EML4-ALK fusion genes in leukocyte and plasma RNA samples from healthy volunteers. Among the 50 clinical samples, 47 ALK-FISH negative samples were also negative. Among 3 ALK-FISH positive samples, 2 cases were detected positive using this method, but another was not detected because of the failure of RNA extraction. Conclusion The proposed qRT-PCR assay for the detection of EML4-ALK fusion genes is rapid, simple, sensitive, and specific, which is deserved to be validated and widely used in clinical settings.

Driven Gene in Patients with Lung Squamous Cell Carcinoma: Analysis of Clinicopathologic Characteristics and Prognosis

背景与目的表皮生长因子受体（epidermal growth factor receptor, EGFR）、间变淋巴瘤激酶（anaplastic lymphoma kinase, ALK）以及KRAS基因是非小细胞肺癌（non-small cell lung cancer, NSCLC）常见的驱动基因。多项临床研究已证实，EGFR敏感突变及棘皮动物微管相关类蛋白4-间变淋巴瘤激酶（echinoderm microtubule-associated protein like 4-anaplastic lymphoma kinase, EML4-ALK）基因重排的晚期肺腺癌患者，一线治疗选择靶向药物优于化疗，其在总缓解率（overall response rate, ORR）、无进展生存期（progression-free survival, PFS）及生活质量方面均有明显优势。然而，在肺鳞癌患者中，目前尚无明确的基因检测位点及靶向药物来指导临床治疗。本研究旨在分析肺鳞癌患者EGFR、ALK以及KRAS基因的突变状态，以及相关的临床病理特征，从而为今后的治疗提供相应指导。方法回顾性分析已行相关驱动基因检测的肺鳞癌患者90例，利用突变扩增阻滞系统（amplification refractory mutation system, ARMS）的方法进行EGFR及KRAS基因检测，荧光原位杂交（fluorescence in situ hybridization, FISH）技术进行ALK基因融合检测。结果90例患者均进行了EGFR及KRAS基因检测，8例患者为EGFR基因突变（8.8%）；2例患者为KRAS基因突变（2.2%）；18例患者通过FISH方法进行了ALK基因融合检测，1例患者存在EML4-ALK基因融合（5.6%）。女性患者的EGFR基因突变率高于男性（P=0.022）。EGFR突变及野生型患者在病理分期（P=0.042）及分化程度（P=0.003）上均有差异。以胸膜为首发转移部位的EGFR突变患者比率高于EGFR野生型患者（P=0.013）。靶向治疗与化疗对EGFR突变患者的PFS无影响（P=0.607）。结论ALK基因融合患者应用靶向治疗后可获得理想疗效。

Echinoderm microtubule-associated protein -like protein 5 in anterior temporal neocortex of patients with intractable epilepsy.

EMAP-like Protein 5 (EML5) is a new echinoderm microtubule-associated protein that is expressed in the central nervous system. The aim of this study was to investigate the expression profile of EML5 in the anterior temporal neocortex of patients presenting with intractable epilepsy (IE). Western blot assays were performed to determine EML5 expression in 36 surgically resected anterior temporal neocortices of patients with IE and eight control tissues. Immunohistochemistry and immunofluorescence were employed to explore protein expression in IE. EML5 was highly expressed in both neurons and glial cells of the anterior temporal neocortex of IE patients, whereas only low levels of EML5 were detected in control brain tissues. Western blotting showed an enhanced expression of EML5 protein in the anterior temporal neocortex of IE (optical density (OD) = 1.8030 ± 0.1335/1.1852 ± 0.2253, P<0.05) compared with normal control tissues. The results demonstrate that highly expressed EML5 in the neurons and glial cells of the cortex of patients with epilepsy is associated with microtubular dysfunction after frequent and recurrent seizures.

Anaplastic Lymphoma Kinase Variants and the Percentage of ALK-Positive Tumor Cells and the Efficacy of Crizotinib in Advanced NSCLC

BackgroundAnaplastic lymphoma kinase (ALK)-positive non–small-cell lung cancer patients exhibited heterogeneous magnitude of response and duration time to criztotinib treatment. This study explored ALK variants and the percentage of ALK-positive cells using fluorescent in situ hybridization (FISH) on clinical efficacy of crizotinib. Patients and MethodsA total of 120 patients with ALK rearrangement who were treated with criztotinib were enrolled. ALK variants were clarified in 61 patients, and ALK percentages were evaluated using FISH in 114 ALK-positive patients. Retrospectively, objective response rate, and progression-free survival (PFS) were evaluated. ResultsA total of 61 patients with specific ALK variants were divided into 3 subgroups, echinoderm microtubule-associated protein like 4 (EML4)-ALK variant 1 (n = 22), EML4-ALK variant 3a/b (n = 18), and other ALK variants (n = 21). Median PFS in the 3 subgroups was 11.0 months (95% confidence interval [CI], 5.5-16.5), 10.9 months (95% CI, 5.9-15.8), 7.4 months (95% CI, 3.2-11.6), respectively, and no significant difference (P = .795) existed among them. The percentage of ALK-positive cells in FISH analysis was weakly correlated with PFS (rs = 0.235; P = .015). Additionally, it was also weakly correlated with best response to crizotinib (rs = 0.288; P = .003). Overall, there were 45, 49, and 26 patients receiving first, second, and third or further-line crizotinib, respectively. Median PFS in the first-line setting (10.5 months; 95% CI, 8.6-12.4) was significantly longer than that in the second-line setting (8.3 months; 95% CI, 4.7-12.0; P = .020). ConclusionAnaplastic lymphoma kinase variants might have no correlation with clinical response to crizotinib. The percentage of ALK-positive cells might correlate with the extent of benefit from crizotinib treatment.

Differences in driver genes between smoking-related and non-smoking-related lung cancer in the Chinese population.

Recently, non-smoking-related lung cancer was classified as an independent disease entity because it is different from tobacco-associated lung cancer. Non-smoking-related lung cancer occurs more often in women than men, and the predominant histological type is adenocarcinoma (ADC) rather than squamous cell carcinoma. Most of the driver gene alterations that have been identified in ADC in never-smokers include epidermal growth factor receptor mutations, KRAS mutations, echinoderm microtubule-associated protein like 4/anaplastic lymphoma kinase fusion, and ROS1 fusion, among others. Meanwhile, significant progress has been made in the treatment of ADC. However, in comparison with ADC, no such available molecular targets exist for smoking-associated lung cancer, for which treatment strategies are limited. Next-generation sequencing has been widely applied to the discovery of more genetic profiles of lung cancers. This review summarizes the differences between smoking-related and non-smoking-related lung cancer as follows: different somatic mutation burdens, C:G→A:T transversions, common and novel driver genes, and treatment strategies. Overall, smoking-related lung cancer is more complicated than non-smoking-related lung cancer. Furthermore, we review the prevalence of driver genes in smoking-associated and non-smoking-associated lung cancers in the Chinese population.