非小细胞肺癌患者EML4-ALK融合基因的检测及其临床病理特征分析

目的:检测EML4-ALK融合基因在非小细胞肺癌(NSCLC)人群中的突变率,并分析其与临床病理特征的关系。方法:采用逆转录定量PCR检测66例NSCLC患者组织标本中EML4-ALK融合基因的突变率;采用DNA扩增后直接测序的方法检测EML4-ALK阳性患者的组织标本中EGFR基因(18~21号外显子)及K-Ras基因(2号外显子)的突变情况。结果:66例NSCLC患者的组织标本中,有5例(7.6%)存在EML4-ALK融合基因阳性,这5例组织标本的EGFR(18~21号外显子)及K-Ras(2号外显子)均为野生型。5例阳性患者中,4例年龄小于总体患者的平均年龄(59±11)岁,占80%(4/5);女性患者4例,占80%(4/5);不吸烟患者3例,占60%(3/5)。EML4-ALK融合基因阳性NSCLC患者均为腺癌,1例NSCLC组织为腺泡样结构,3例组织为印戒细胞样结构,4例伴有胞内或胞外黏液。结论:EML4-ALK融合基因阳性NSCLC多见于年轻女性腺癌患者,多为伴有黏液产生的印戒细胞样结构,不同时合并EGFR和KRas突变。     

基于CRISPR/Cas13a的EML4-ALK融合基因检测方法的建立

[目的]构建基于CRISPR/Cas13a蛋白的SHERLOCK技术平台，并将其应用于肺癌EML4-ALK融合基因的检测。[方法]构建EML4-ALK融合基因的质粒标准品；利用RPA和PCR技术分别扩增目的基因后进行SHERLOCK检测并比对结果，比较基于RPA和PCR扩增的SHERLOCK检测EML4-ALK融合基因的灵敏度，评估SHERLOCK检测目的基因及多种非目标基因的特异性。[结果]成功构建EML4-ALK融合基因表达质粒标准品；基于RPA和PCR扩增的SHERLOCK检测EML4-ALK融合基因的相对荧光值无显著差异，最低检测下限为10 copies/μL,且在5 min内荧光达到平台期；SHERLOCK技术仅对EML4-ALK融合基因有阳性检测信号，对EML4、ALK、EGFR T790M和EGFR L858R基因无阳性检测信号。[结论]基于SHERLOCK技术的EML4-ALK融合基因检测方法具有特异性强、灵敏度高、检测时间短等优势，有望为肺癌EML4-ALK融合基因的早期诊断提供重要依据。     

外泌体介导长链非编码RNA H19促进肝癌细胞增殖与转移

外泌体是由细胞分泌的直径为30～150 nm的小囊泡,含有丰富的mRNA、microRNA和长链非编码RNA(lncRNA)。目前,大多数外泌体研究都集中在mRNA和microRNA,而对lncRNA的生物学功能并不十分清楚。研究表明,肿瘤细胞外泌体 lncRNA H19在肿瘤细胞的增殖、迁移和侵袭中发挥了重要作用。本研究将筛选到的lncRNA H19高表达的肝癌细胞HCCLM3,分别收集其高表达lncRNA H19的外泌体和其下调lncRNA H19表达后的外泌体。然后,将收集到的外泌体分别添加到lncRNA H19低表达的肝癌细胞Hep3B和HepG2孵育液中。孵育24 h后,检测其对肿瘤细胞的增殖、迁移和侵袭能力的影响。结果显示,肝癌细胞HCCLM3可分泌大量的外泌体,且能被其他肿瘤细胞大量摄取;与下调lncRNA H19表达的外泌体相比,lncRNA H19高表达的外泌体能显著增强Hep3B和HepG2细胞的增殖、迁移和侵袭能力。而这一作用可通过激活PI3K/AKT/mTOR通路实现。上述结果表明,lncRNA H19高表达的肝癌细胞以外泌体方式,增强邻近肝癌细胞的增殖、迁移和侵袭能力,促进肝癌的发生与发展。     

外泌体介导的长链非编码RNA H19促进肝癌细胞的增殖与转移

外泌体是由细胞分泌的直径为30～150 nm的小囊泡,含有丰富的mRNA、microRNA和长链非编码RNA(lncRNA)。目前,大多数外泌体研究都集中在mRNA和microRNA,而对lncRNA的生物学功能并不十分清楚。研究表明,肿瘤细胞外泌体lncRNA H19在肿瘤细胞的增殖、迁移和侵袭中发挥了重要作用。本研究将筛选到的lncRNA H19高表达的肝癌细胞HCCLM3,分别收集其高表达lncRNA H19的外泌体和其下调lncRNA H19表达后的外泌体。然后,将收集到的外泌体分别添加到lncRNA H19低表达的肝癌细胞Hep3B和HepG2孵育液中。孵育24 h后,检测其对肿瘤细胞的增殖、迁移和侵袭能力的影响。结果显示,肝癌细胞HCCLM3可分泌大量的外泌体,且能被其他肿瘤细胞大量摄取;与下调lncRNA H19表达的外泌体相比,lncRNA H19高表达的外泌体能显著增强Hep3B和HepG2细胞的增殖、迁移和侵袭能力。而这一作用可通过激活PI3K/AKT/mTOR通路实现。上述结果表明,lncRNA H19高表达的肝癌细胞以外泌体方式,增强邻近肝癌细胞的增殖、迁移和侵袭能力,促进肝癌的发生与发展。     

间充质干细胞外泌体中的RNA与蛋白质

间充质干细胞(mesenchymal stem cell,MSC)外泌体是MSC中的多泡小体与细胞膜融合时分泌到细胞外环境中的50～200 nm大小的细胞外囊泡。MSC外泌体含有TSG101、CD9和CD81等典型蛋白质与多种RNA。人们逐渐认识到外泌体是通过传递其蛋白质和RNA等内容物到受体细胞发挥作用的。因此,MSC外泌体的治疗潜能可能是因为它含有特殊的蛋白质或RNA。该文对外泌体蛋白质和RNA的作用机制进行探究,并提出MSC外泌体很可能通过蛋白质而不是RNA发挥效应。     

外泌体与乳腺癌关系的研究进展

外泌体是细胞经过"内吞-融合-外排"等一系列调控过程而形成的细胞外纳米级小囊泡。外泌体通过其携带的蛋白或RNA影响受体基因调控网络或表观遗传重组,进而调控细胞的生理过程。研究表明,乳腺癌细胞自身分泌的外泌体和外界细胞分泌的外泌体在乳腺癌发生、发展过程中的细胞迁移、细胞分化和免疫应答等方面发挥重要作用。本文对外泌体与乳腺癌关系的研究进展进行综述。     

外泌体及其抗肿瘤作用机制

外泌体是由细胞分泌到胞外的囊泡状小体,体内多种细胞可以分泌外泌体,来源于树突状细胞（DC）和肿瘤细胞的外泌体表面表达MHC分子和抗原肽,体内外实验证明其具有抗肿瘤的作用,因此外泌体作为抗肿瘤疫苗被广泛研究。该文介绍外泌体的发现、蛋白质组成、体内抗肿瘤的机制,以及DC与肿瘤来源的外泌体的基础及临床研究。     

外泌体在白血病中的重要调控作用

目前,白血病复发是患者死亡的主要原因之一。肿瘤细胞和微环境的相互作用,以及隐匿在骨髓中的肿瘤干细胞,促进了白血病的复发和向淋巴组织的转移,因此白血病的治疗、转移和复发问题受到广泛关注。外泌体是由绝大多数细胞分泌的双层脂质膜囊泡,可以调控细胞间的交流和信息传递。在白血病细胞、基质细胞和内皮细胞之间的相互联系中都涉及到外泌体,白血病细胞来源的外泌体存在于白血病患者的血浆中,能把其携带的白血病相关抗原及微小RNA呈递给靶细胞,促进白血病肿瘤细胞的增殖,有助于肿瘤细胞实现免疫逃避,保护白血病细胞抵抗化疗药物导致的细胞毒性作用,促进血管生成及肿瘤细胞的迁移。因此,外泌体与白血病的转移、治疗及预后密切相关,可以用来检测和监测白血病的进展。本文综述了外泌体的来源、形成与分泌机制,以及外泌体在白血病发生前、发展中、预后和免疫治疗中所扮演的重要角色。     

外泌体环状RNA在泌尿系统肿瘤中的研究进展

外泌体是一种包含了复杂RNA和蛋白质的膜性囊泡，其主要来源于细胞内溶酶体微粒内陷形成的多囊泡体，经多囊泡体外膜与细胞膜融合后释放到胞外基质中。外泌体在肿瘤微环境中介导细胞间通讯，其功能取决于来源的细胞类型。环状RNA是一类由前体mRNA反向剪接生成的非编码RNA，在外泌体中富集且稳定表达。外泌体环状RNA在疾病中发挥了重要的调控作用，其作为肿瘤标志物和治疗靶点的临床应用前景与价值现已成为研究热点。本文就外泌体环状RNA在泌尿系统肿瘤中的研究进展作一综述。     

支原体污染增加人肿瘤细胞外泌体生成并改变其小RNA表达模式

目的:研究支原体污染对肿瘤细胞外泌体生成的影响,探讨支原体导致的外泌体量和质的变化。方法:应用荧光显微镜及荧光共振能量转移观察支原体污染导致的胞内膜脂质向胞膜再分布及跨细胞传输变化,应用western blotting检测污染细胞上清中外泌体标记物CD81蛋白含量,应用芯片检测污染细胞上清外泌体中小RNA的表达变化模式。结果:支原体污染导致Rab11和胞膜脂质染料信号强度上升2倍以上,提示胞内膜脂质向胞膜再分布增强;支原体污染导致Di O和Di I双染细胞胞内膜泡的FRET指数上升4倍,提示膜脂质由支原体污染细胞向未污染细胞的跨细胞传输增强;污染细胞的上清中外泌体标志物CD81明显升高;污染导致外泌体内小RNA表达模式改变。结论:体外实验显示支原体污染可造成外泌体质和量的改变。     

EGFR突变与EML4-ALK融合共存的非小细胞肺癌的临床病理特征及治疗分析

目的:探讨表皮生长因子受体(EGFR)基因突变与棘皮动物微管相关样蛋白4与间变性淋巴瘤激酶(EML4-ALK)融合基因共存(以下简称双基因异常)的非小细胞肺癌的临床病理特征及治疗策略。方法:回顾性收集并分析2012年1月至2016年12月我院收治的EGFR突变与EML4-ALK融合基因共存的非小细胞肺癌患者的临床资料及病理特点。结果:11例双突变非小细胞肺癌占医院同期入院非小细胞肺癌患者的0.68%(11/1620);男性6例,女性5例;年龄23-70岁,平均年龄51.6岁;11例患者均不吸烟;腺癌9例,肉瘤样癌2例;临床分期,ⅠA期3例,ⅡB期1例,ⅢA期1例,ⅢB期1例,ⅠV期5例;6例行手术治疗,4例使用传统化疗,最好疗效为稳定(SD),最长无进展生存期(PFS)为6月;5例患者使用表皮生长因子酪氨酸激酶抑制剂(EGFR-TKⅠ)治疗,使用EGFR-TKⅠ最好疗效为部分缓解(PR),PFS为3-23月,中位PFS为9月;截止2017年12月,死亡4例,11例患者的生存时间为1-67月,中位存活时间为21月。结论:EGFR基因突变与EML4-ALK融合基因共存型非小细胞肺癌临床少见,多见于不吸烟或少吸烟的肺腺癌患者,双基因异常的非小细胞肺癌的靶向药物的治疗缺乏统一性,有待进一步研究,基于EGFR及EML4-ALK的磷酸化水平或肿瘤突变负荷选择靶向药物的个体化精准治疗是非常重要的。     

Clinicopathologic Features of Patients with Non-Small Cell Lung Cancer Harboring the EML4-ALK Fusion Gene: A Meta-Analysis

Background

The frequencies of EML4-ALK fusion gene in non-small cell lung cancer (NSCLC) with different clinicopathologic features described by previous studies are inconsistent. The key demographic and pathologic features associated with EML4-ALK fusion gene have not been definitively established. This meta-analysis was conducted to compare the frequency of the EML4-ALK fusion gene in patients with different clinicopathologic features and to identify an enriched population of patients with NSCLC harboring EML4-ALK fusion gene.

Methods

The Pubmed and Embase databases for all studies on EML4-ALK fusion gene in NSCLC patients were searched up to July 2014. A criteria list and exclusion criteria were established to screen the studies. The frequency of the EML4-ALK fusion gene and the clinicopathologic features, including smoking status, pathologic type, gender, and EGFR status were abstracted.

Results

Seventeen articles consisting of 4511 NSCLC cases were included in this meta-analysis. A significant lower EML4-ALK fusion gene positive rate was associated with smokers (pooled OR = 0.40, 95% CI = 0.30–0.54, P<0.00001). A significantly higher EML4-ALK fusion gene positivity rate was associated with adenocarcinomas (pooled OR = 2.53, 95% CI = 1.66–3.86, P<0.0001) and female (pooled OR = 0.61, 95% CI = 0.41–0.90, P = 0.01). We found that a significantly lower EML4-ALK fusion gene positivity rate was associated with EGFR mutation (pooled OR = 0.07, 95% CI = 0.03–0.19, P<0.00001). No publication bias was observed in any meta-analysis (all P value of Egger''s test >0.05); however, because of the small sample size, no results were in the meta-analysis regarding EGFR gene status.

Conclusion

This meta-analysis revealed that the EML4-ALK fusion gene is highly correlated with a never/light smoking history, female and the pathologic type of adenocarcinoma, and is largely mutually exclusive of EGFR.     

胶质瘤来源外泌体通过高迁移率族蛋白B1促进胶质瘤干细胞形成

摘要 目的：研究胶质瘤来源外泌体中高迁移率族蛋白B1（HMGB1）对胶质瘤干细胞形成的影响及其意义。方法：使用外泌体提取试剂盒提取原代胶质母细胞瘤来源外泌体,通过透射电子显微镜、纳米粒度电位仪和Western blotting对外泌体进行鉴定;采用Western blotting检测外泌体中HMGB1的表达量;通过qRT-PCR、Western blotting、克隆球计数检测外泌体对胶质瘤干细胞形成的影响;siRNA敲低HMGB1的表达水平,并通过qRT-PCR、Western blotting、克隆球计数检测外泌体中HMGB1对胶质瘤干细胞形成的影响。结果：原代胶质瘤细胞可以分泌外泌体到肿瘤微环境并且外泌体中存在HMGB1;原代胶质瘤细胞来源外泌体可以上调邻近胶质瘤细胞干性相关分子CD133、OCT4、NANOG、SOX2的表达并促进干细胞克隆球的形成;通过siRNA敲低原代胶质瘤细胞HMGB1的表达后,外泌体中HMGB1的含量降低并且外泌体促进胶质瘤干细胞形成的作用减弱。结论：胶质瘤细胞来源外泌体可以通过HMGB1促进胶质瘤干细胞的形成。     

NK92-CD16 cells are cytotoxic to non-small cell lung cancer cell lines that have acquired resistance to tyrosine kinase inhibitors

BackgroundTreatment with tyrosine kinase inhibitors (TKIs) has improved the outcomes for patients with non-small cell lung cancer (NSCLC) harboring targetable driver mutations. However, acquired resistance to TKIs invariably develops within approximately 1 year of treatment by various mechanisms, including gatekeeper mutations, alternative pathway activation and histological transformations. Because immunotherapy is an option for patients with drug-resistant cancers, we generated several TKI-resistant NSCLC cell lines in vitro, and then evaluated the cytotoxicity of NK92-CD16 cells to these resistant cells.Materials and MethodsTKI-resistant NSCLC cells (H3122CR1, H3122LR1, H3122CR1LR1, PC-9GR, PC-9ER, EBC-CR1 and EBC-CR2) were established from NCI-H3122 (EML4-ALK fusion), PC-9 (EGFR exon19 deletion) and EBC-1 (MET amplification) after continuous exposure to crizotinib, ceritinib, gefitinib, erlotinib and capmatinib. Expression of ligands for natural killer (NK) cell receptors and total EGFR were analyzed using flow cytometry. NK cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC) using anti-EGFR monoclonal antibody (mAb) cetuximab were measured using NK92-CD16 as effectors and detected using the ⁵¹Chromium-release assay.ResultsWe found that NK92-CD16 cells preferentially killed TKI-resistant NSCLC cells when compared with their parental NSCLC cells. Mechanistically, intracellular adhesion molecule 1 (ICAM-1) was up-regulated in the TKI-resistant NSCLC cells and patients’ tumors, and the ICAM-1 up-regulated cancer cells lines were less susceptible to NK cytotoxicity by blocking ICAM-1. Moreover, NK92-CD16 cell-induced cytotoxicity toward TKI-resistant NSCLC cells was enhanced in the presence of cetuximab, an EGFR-targeting mAb.ConclusionThese data suggest that combinational treatment with NK cell–based immunotherapy and cetuximab may be promising for patients with TKI-resistant NSCLC.     

In Vitro Drug Sensitivity Tests to Predict Molecular Target Drug Responses in Surgically Resected Lung Cancer

BackgroundEpidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) and anaplastic lymphoma kinase (ALK) inhibitors have dramatically changed the strategy of medical treatment of lung cancer. Patients should be screened for the presence of the EGFR mutation or echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusion gene prior to chemotherapy to predict their clinical response. The succinate dehydrogenase inhibition (SDI) test and collagen gel droplet embedded culture drug sensitivity test (CD-DST) are established in vitro drug sensitivity tests, which may predict the sensitivity of patients to cytotoxic anticancer drugs. We applied in vitro drug sensitivity tests for cyclopedic prediction of clinical responses to different molecular targeting drugs.MethodsThe growth inhibitory effects of erlotinib and crizotinib were confirmed for lung cancer cell lines using SDI and CD-DST. The sensitivity of 35 cases of surgically resected lung cancer to erlotinib was examined using SDI or CD-DST, and compared with EGFR mutation status.ResultsHCC827 (Exon19: E746-A750 del) and H3122 (EML4-ALK) cells were inhibited by lower concentrations of erlotinib and crizotinib, respectively than A549, H460, and H1975 (L858R+T790M) cells were. The viability of the surgically resected lung cancer was 60.0 ± 9.8 and 86.8 ± 13.9% in EGFR-mutants vs. wild types in the SDI (p = 0.0003). The cell viability was 33.5 ± 21.2 and 79.0 ± 18.6% in EGFR mutants vs. wild-type cases (p = 0.026) in CD-DST.ConclusionsIn vitro drug sensitivity evaluated by either SDI or CD-DST correlated with EGFR gene status. Therefore, SDI and CD-DST may be useful predictors of potential clinical responses to the molecular anticancer drugs, cyclopedically.     

TIM-3 shuttled by MV3 cells-secreted exosomes inhibits CD4+ T cell immune function and induces macrophage M2 polarization to promote the growth and metastasis of melanoma cells

This study is sought to determine the physiological mechanisms by which exosomes-encapsulated TIM-3 derived from melanoma cells might mediate CD4⁺ T cell immune function and macrophage M2 polarization in melanoma. Initially, exosomes were isolated from the human skin-derived melanoma cell line MV3for analysis of TIM-3 expression pattern. Next, the exosomes sourced from MV3 cells manipulated with sh-TIM-3 were co-incubated with CD4⁺ T cells to detect CD4⁺ T cell proliferation and MV3 cell migration and invasion, to observe the macrophage M2 polarization, and to determine levels of several EMT-related factors. Finally, melanoma nude mouse models were established to study the in vivo modulatory effects of TIM-3 from MV3 cells-derived exosomes. MV3 cells-derived exosomes inhibited CD4⁺ T cell immune function and promoted macrophage M2 polarization in melanoma. Our results revealed the abundance of TIM-3 in MV3 cells-derived exosomes. Of importance, silencing of TIM-3 shuttled by MV3 cells-derived exosomes improved CD4⁺ T cell immune function and inhibited macrophage M2 polarization to attenuate the growth and metastasis of melanoma cells. Collectively, MV3 cells-derived exosomes-loaded TIM-3 suppressed CD4⁺ T cell immune function and induced macrophage M2 polarization to improve occurrence and development of melanoma, therefore providing us with a potential therapeutic target for effectively combating melanoma.     

Genomic Aberrations in Crizotinib Resistant Lung Adenocarcinoma Samples Identified by Transcriptome Sequencing

ALK-break positive non-small cell lung cancer (NSCLC) patients initially respond to crizotinib, but resistance occurs inevitably. In this study we aimed to identify fusion genes in crizotinib resistant tumor samples. Re-biopsies of three patients were subjected to paired-end RNA sequencing to identify fusion genes using deFuse and EricScript. The IGV browser was used to determine presence of known resistance-associated mutations. Sanger sequencing was used to validate fusion genes and digital droplet PCR to validate mutations. ALK fusion genes were detected in all three patients with EML4 being the fusion partner. One patient had no additional fusion genes. Another patient had one additional fusion gene, but without a predicted open reading frame (ORF). The third patient had three additional fusion genes, of which two were derived from the same chromosomal region as the EML4-ALK. A predicted ORF was identified only in the CLIP4-VSNL1 fusion product. The fusion genes validated in the post-treatment sample were also present in the biopsy before crizotinib. ALK mutations (p.C1156Y and p.G1269A) detected in the re-biopsies of two patients, were not detected in pre-treatment biopsies. In conclusion, fusion genes identified in our study are unlikely to be involved in crizotinib resistance based on presence in pre-treatment biopsies. The detection of ALK mutations in post-treatment tumor samples of two patients underlines their role in crizotinib resistance.     

Clinicopathological Characteristics of Patients with Non-Small-Cell Lung Cancer Who Harbor EML4-ALK Fusion Gene: A Meta-Analysis

BackgroundA novel fusion gene of echinoderm microtubule-associated protein-like 4 (EML4) and anaplastic lymphoma kinase (ALK) has been recently identified in non-small-cell lung cancers (NSCLCs). Patients with the EML4-ALK fusion gene demonstrate unique clinicopathological and physiological characteristics. Here we present a meta-analysis of large-scale studies to evaluate the clinicopathological characteristics of NSCLC patients harboring the EML4-ALK fusion gene.MethodsBoth English and Chinese databases were systematically used to search the materials of the clinicopathological characteristics of patients with NSCLC harboring the EML4-ALK fusion gene. Pooled relative risk (RR) estimates and the 95% confidence intervals (95% CI) were calculated with the fixed or random effect model. Publication bias and chi-square test were also calculated.Results27 retrospective studies were included in our meta-analysis. These studies included a total of 6950 patients. The incidence rate of EML4-ALK fusion in NSCLC patients was found to be 6.8% (472/6950). The correlation of the EML4-ALK fusion gene and clinicopathological characteristics of NSCLC patients demonstrated a significant difference in smoking status, histological types, stage, and ethnic characteristics. The positive rate of the EML4-ALK fusion gene expression in females were slightly higher than that in males, but not significantly (P = 0.52). In addition, the EML4-ALK fusion gene was mutually exclusive of the EGFR and KRAS mutation genes (P = 0.00).ConclusionOur pooled analysis revealed that the EML4-ALK fusion gene was observed predominantly in adenocarcinoma, non-smoking and NSCLC patients, especially those diagnosed in the advanced clinical stage of NSCLC. Additionally, the EML4-ALK fusion gene was exclusive of the EGFR and KRAS mutation genes. We surmise that IHC assay is a valuable tool for the prescreening of patients with ALK fusion gene in clinical practice, and FISH assay can be performed as a confirmation method. These insights might be helpful in guiding the appropriate molecular target therapy for NSCLC.