Circulating tumor cells with epithelial-mesenchymal transition markers as potential biomarkers for the diagnosis of lung cancer

BACKGROUNDCirculating tumor cells (CTCs) can be clustered into three subtypes according to epithelial-mesenchymal transition (EMT) markers: CTCs with epithelial markers (E-CTCs), CTCs with mesenchymal markers (M-CTCs), and CTCs with both markers (E&M-CTCs). CTC detection has clinical implications in the diagnosis of lung cancer (LC). AIMTo clarify the diagnostic value of CTCs categorized by EMT markers in LC.METHODSThe study included 106 patients with lung adenocarcinoma, including 42 ground-glass opacities (GGO) and 64 solid lesions, who underwent surgery between July 2015 and December 2019. Eleven patients with benign tumors and seventeen healthy controls were included. CTCs in peripheral blood and associated EMT markers were detected preoperatively using the CanPatrol^TM technique. The diagnostic power of CTCs for discriminating LC cases from controls was analyzed by the receiver operating characteristic (ROC) curve. The CytoploRare technique was used in 20 cases and 18 controls for validation, and Kappa values were calculated to evaluate consistency between techniques.RESULTSOf the 106 LC cases, 94 (89.6%) had at least one CTC. CTCs were detectable in 35 (83.3%) of 42 GGO cases. Total CTCs and E&M-CTCs were significantly more frequent in LC cases than in benign or healthy controls. The proportion of M-CTCs plus E&M-CTCs increased gradually from healthy controls, to benign controls, to LC cases. The area under the ROC curve of total CTCs and E&M-CTCs was > 0.8 and > 10.75, respectively. The combined sensitivity of total-CTCs and E&M-CTCs was 85.85% for LC patients (80.95% for GGO patients) and the specificity was 78.57%. The Kappa value was 0.415, indicating relative consistency between CanPatrol^TM and CytoploRare. CONCLUSIONCTC detection is valuable for distinguishing LC from controls, and particularly E&M-CTC detection warrants further study.     

循环肿瘤细胞与肿瘤标志物在肺癌诊断中的价值比较

目的探讨循环肿瘤细胞(CTCs)检测在肺癌中的诊断价值。方法选取35例肺癌患者为肺癌组,以同期入院的非结核肺良性疾病患者22例及健康体检者26例为对照组,分别检测两组患者外周血CTCs及传统血清肿瘤标志物的表达水平,并使用灵敏度、特异度及ROC曲线对CTCs在肺癌中的诊断价值进行评价。结果肺癌组CTCs的检出率为77.1%,显著高于对照组CTCs检出率(10.4%),差异具有统计学意义(P<0.05);CTCs诊断肺癌的灵敏度、特异度、ROC曲线下面积分别为77.1%、89.6%、0.865,均高于传统血清肿瘤标志物NSE(42.9%、77.1%、0.591),CA125(34.3%、87.5%、0.706),CA153(11.4%、89.5%、0.530),CYFRA21-1(62.9%、64.6%、0.674),SCCA(22.9%、66.7%、0.336);而CEA的特异度(93.8%)虽高于CTCs,但其灵敏度(45.7%)及ROC曲线下面积(0.793)均低于CTCs。结论CTCs检测可用于肺癌的诊断,且与传统血清肿瘤标志物相比具有更高的诊断价值。     

Clinical use of novel urine and blood based prostate cancer biomarkers: A review

In the era of upcoming techniques for molecular profiling, breakthroughs led to new discoveries in the field of prostate cancer (PCa) biomarkers. Since the early 1990s a tremendous increase in PCa incidence is seen, dedicated to the introduction of prostate specific antigen (PSA) testing. However, due to its lack of specificity many men undergo unnecessary biopsies, resulting in a rising incidence of clinically insignificant PCa. To overcome this drawback, cancer specific biomarkers are needed to identify patients who are at high risk of harbouring PCa and to distinguish patients with aggressive disease from patients with insignificant cancer. The most non-invasive, easy to obtain substrate for biomarker measurement is urine. The most promising markers to date are PCA3 and TMPRSS2–ERG. Both markers demonstrate to have a higher specificity and diagnostic accuracy for PCa outcome compared to serum PSA. This might better predict the presence of PCa and therefore reduce the number of unnecessary biopsies. Combining both markers in a panel might result in an even higher diagnostic accuracy, given the heterogeneity of the disease. In PCa management, circulating tumour cells (CTCs) detected in the blood seem a promising tool to predict treatment response and survival benefit. Although results appear to be encouraging, the biggest challenge about new markers in PCa is to validate them in large clinical trials and subsequently implement these markers into clinical practice. In this review we discuss the clinical usefulness of novel, non-invasive tests in PCa management.     

全新靶向小分子核酸适配体特异性检测肺癌患者血清标志物的应用研究

目的以纳米磁珠为筛选介质,利用富集配体系统进化技术(SELEX)从肺癌患者血清中筛选得到高特异性、强亲和力的肺癌血清核酸适配体,并进一步构建基于核酸适配体的肺癌血清标志物检测方法。方法以羧基官能团修饰的磁性纳米颗粒为载体、肺癌患者血清为筛选正向靶标、正常人血清为负向靶标,利用SELEX技术从含有77个碱基的随机ssDNA文库中筛选出与肺癌血清特异性结合的核酸适配体,流式细胞术监测筛选进程;当筛选达到饱和时对富集文库进行克隆测序并鉴定克隆的靶标结合特异性。选择性能最优的核酸适配体用于肺癌患者血清标志物的检测,通过50例肺癌患者血清和50例正常人血清鉴定该方法的特异性与敏感度。结果SELEX筛选进行到第9轮时,富集达到最大化。对筛选文库克隆测序,发现seq24和seq85的结合特异性更优,与正常人血清和白蛋白几乎不结合。核酸适配体seq24的Kd值为98.56 nmol/L,seq85的Kd值为128.69 nmol/L;将构建的基于核酸适配体seq24的肺癌患者血清检测方法与目前临床应用的方法比较,两者结果一致,阳性检出率为100%;此外,所构建的方法可区分早期和晚期肺癌患者。结论核酸适配体seq24能以高特异性和亲和力识别肺癌患者血清,以其为基础的全新检测体系可为肺癌的早期诊断提供新方法。     

Circulating tumor cells: Advances in isolation and analysis,and challenges for clinical applications

Circulating tumor cells (CTCs) are rare cancer cells released from tumors into the bloodstream that are thought to have a key role in cancer metastasis. The presence of CTCs has been associated with worse prognosis in several major cancer types, including breast, prostate and colorectal cancer. There is considerable interest in CTC research and technologies for their potential use as cancer biomarkers that may enhance cancer diagnosis and prognosis, facilitate drug development, and improve the treatment of cancer patients. This review provides an update on recent progress in CTC isolation and molecular characterization technologies. Furthermore, the review covers significant advances and limitations in the clinical applications of CTC-based assays for cancer prognosis, response to anti-cancer therapies, and exploratory studies in biomarkers predictive of sensitivity and resistance to cancer therapies.     

Challenges for CTC-based liquid biopsies: low CTC frequency and diagnostic leukapheresis as a potential solution

Circulating tumor cells (CTCs) are very attractive surrogate markers for systemic cancer. Currently, major efforts are being made to use these rare cells in the sense of a liquid biopsy to gain molecular information for rational therapeutic decision-making. The advancements in molecular analyses of CTCs down to the single-cell level have been significant in recent years and some applications are ready to be used in clinical studies. As discussed in this review, a major challenge for translating such molecular CTC-based assays into the clinic is the extremely low frequency of CTCs and the associated problems of their reliable detection and isolation. A potential solution to overcome the low CTC frequency is the recently introduced diagnostic leukapheresis that permits screening of liters of blood. Discussed here are the challenges as well as the current efforts implementing this method into clinical workflows to realize more reliable liquid biopsies.     

肺癌肿瘤标志物神经元特异性烯醇化酶抗原适配子的筛选及其应用

目的提出一种基于适配子快速检测肺癌肿瘤标志物的方法。方法利用指数级富集配体的系统进化技术筛选其高特异性适配子,通过流式细胞术检测亲和力,最终选出一条高亲和力、强特异性的适配子。枸橼酸钠还原法制备纳米金颗粒。建立基于免标记纳米金和适配子的分析方法,并对其方法进行考察。结果通过 6 轮筛选得到三条 NSE 的高特异性、强亲和力的适配子,其中,a 适配子特异性最高。同时利用筛选得到的肺癌肿瘤标志物 a 适配子和纳米金颗粒建立一种快速检测 NSE 抗原的方法。结论该方法具有较好的准确度,为肺癌的早期检测提供了一种新的手段。     

Classifying circulating tumor cells to monitor cancer progression

Introduction: The term ‘liquid biopsy’ refers to molecular analysis of a tumor’s genetic features based on circulating genetic material in the peripheral blood derived from circulating tumor cells (CTCs), circulating tumor DNA (ctDNA) and circulating miRNAs, and has emerged as a minimally invasive tool in early cancer diagnosis and disease monitoring. CTCs are believed to originate from the primary tumor and obtain genetic heterogeneity during evolution.

Areas covered: The presence of CTCs has been associated with poor clinical outcome in patients with metastatic breast cancer, lung cancer, colorectal cancer and prostate cancer. In addition, the detection of CTCs in patients with early breast cancer has been shown to represent an independent prognostic factor associated with an unfavorable clinical outcome. Moreover, the longitudinal evaluation of CTCs in patients with early breast cancer may reveal the presence of chemo- and hormone-therapy resistant CTCs which are associated with an increased risk for disease relapse and disease-related death.

Expert commentary: The molecular characterization of CTCs may provide an important tool for the monitoring and the evaluation of treatment efficacy in patients with different tumor types such as breast, prostate, colon, and non-small cell lung cancer.     

4项肿瘤标志物在肺癌诊断中的应用价值

目的探讨肿瘤标志物胃泌素释放肽前体(ProGRP)、神经元特异性烯醇化酶(NSE)、细胞角蛋白19的可溶性片段(CYFRA21-1)和鳞状细胞癌抗原(SCC)在肺癌诊断中的临床价值。方法选择151例肺癌及肺部良性病变患者为研究对象,其中小细胞肺癌(SCLC)43例,非小细胞肺癌(NSCLC)68例(包括腺癌35例、鳞癌33例),肺部良性病变患者40例,另选取健康体检者40例纳入健康对照组。采用罗氏电化学发光仪E601检测所有研究对象血清中的ProGRP、NSE、CYFRA21-1和SCC水平。结果 SCLC组的ProGRP和NSE水平均明显高于NSCLC-腺癌组、NSCLC-鳞癌组及肺部良性病变组,而NSCLC-腺癌组和NSCLC-鳞癌组的CYFRA21-1和SCC水平均明显高于SCLC组及肺部良性病变组,差异均有统计学意义(P<0.05)。在单项指标中,ProGRP在SCLC中的灵敏度和特异度最高,分别为77.2%和91.1%;CYFRA21-1在NSCLC-腺癌和NSCLC-鳞癌中的灵敏度和特异度较高,分别为65.9%和90.1%,以及71.5%和91.2%;在联合检测中,ProGRP+NSE在SCLC中的灵敏度和特异度分别为92.5%和83.6%;CYFRA21-1+SCC对NSCLC-腺癌和NSCLC-鳞癌的灵敏度在83.0%以上,特异度在85.0%以上。结论 ProGRP与NSE联合检测诊断SCLC的价值优于单项肿瘤标志物的检测;CYFRA21-1与SCC联合检测对NSCLC-腺癌和NSCLC-鳞癌有较高的灵敏度和特异度,这些肿瘤标志物诊断和筛查肺癌的潜力值得进一步研究。     

非小细胞肺癌循环肿瘤细胞计数的临床应用价值

[目的]评价非小细胞肺癌（NSCLC）患者的外周静脉血中循环肿瘤细胞（CTCs）计数的临床应用价值.[方法]对10例健康成人,10例肺部良性肿块患者和49例NSCLC患者进行CTCs计数.分析CTCs阳性率与年龄、性别、吸烟史、病理类型、细胞分化程度和临床分期的关系.以CTCs计数的中位数12个/7.5 mL为标准将Ⅳ期NSCLC患者分为两组,随访8个月,比较其平均生存时间.[结果]健康成人和良性患者外周血中未发现CTCs,在NSCLC患者中,随着TNM分期的增高,CTCs数目有增多的趋势.阳性率与病理类型、细胞分化程度和临床分期有密切关系（P〈0.05）.Ⅳ期NSCLC患者中CTCs≥12个/7.5 mL组的平均生存时间小于CTCs计数〈12个/7.5 mL组（P=0.040）.[结论]NSCLC的CTCs计数反映患者的疾病状态.其有助于发现隐形微转移、重新确定临床分期及指导患者术后的个体化治疗.晚期NSCLC患者中CTCs计数对其生存期有较好的预测作用.     

Regulation of oncogenic genes by MicroRNAs and pseudogenes in human lung cancer

Lung cancer is one of the most common mortal cancer types both for men and women. Several different biomarkers have been analyzed to reveal lung cancer prognosis pathways for developing efficient therapeutics and diagnostic agents. microRNAs (miRNAs) and pseudogenes are critical biomarkers in lung cancer and alteration of their expression levels has been identified in each step of lung cancer tumorigenesis. miRNAs and pseudogenes are crucial gene regulators in normal cells as well as in lung cancer cells, and they have both oncogenic and tumor-suppressive roles in lung cancer tumorigenesis. In this study, we have determined the relationship between lung cancer related oncogenes and miRNAs along with pseudogenes in lung cancer, and the results indicate their potential as biological markers for diagnostic and therapeutic purposes.     

外周血循环肿瘤细胞检测在非小细胞肺癌治疗的疗效判断及预后预测中的作用

目的探究外周血循环肿瘤细胞(CTCs)的检测在非小细胞肺癌(NSCLC)治疗的疗效判断及预后预测中的作用。方法收集本院收治的NSCLC患者64例,检测其治疗前、治疗后3周及3个月的外周血CTCs数量,患者均随访1年,对其局部转移复发率进行统计。探讨CTCs及其变化与NSCLC疗效、预后之间的关系。结果 CTCs阴性患者的TNM总分期、是否远处转移的构成比与CTCs阳性患者有显著差异(P<0.05)。治疗后3周及治疗后3个月,NSCLC患者的CTCs数目及阳性率均低于治疗前(P<0.05)。治疗后3个月,CTCs阴性患者的RR高于CTCs阳性患者(P<0.05)。治疗后随访期间,CTCs阴性患者局部转移复发率低于CTCs阳性患者(P<0.05)。结论外周血CTCs检测可用于NSCLC的疗效评价,且对患者具有一定的预后预测价值。     

Potentially useful biomarkers for the diagnosis, treatment and prognosis of lung cancer.

Lung cancer ranks top in both incidence and mortality in most part of the world. Scientists strive to explore biomarkers and their possible role in the diagnosis, treatment and prognosis of lung cancer. The ultimate goal is to discover biomarkers that can be tested in clinical trials and finally applied to patient care. Highly elevated concentrations of cytokeratin 19 fragment, tissue polypeptide antigen and squamous cell carcinoma antigen in non-small cell lung cancer particularly for squamous cell carcinoma, carcinoembryonic antigen and cancer antigen 125 in adenocarcinoma or non-small cell lung cancer, as well as progastrin-releasing peptide and neuron specific enolase in small cell lung cancer are suggestive biomarkers for the malignancy. Despite extensive studies, most results still remain controversial. Even with the report of high percent sensitivity and specificity, validation by clinical trials in large cohorts of patients is necessary before the cancer-related phenotypes can be translated into the clinic as reliable biomarkers. Nevertheless, identifications of biomarkers are leading to more understanding of the molecular pathways involved in lung cancer. It is hoped that understanding the connections between cellular pathways will help to reduce the suffering and loss of life caused by the lethal disease. This article summarizes the pre-clinical and translational researches against lung cancer in relation to biomarker discovery and validation. It is intended for policy makers, researchers, clinicians and other health professionals, offering a variety of useful biomarkers and updated data of clinical trials for lung cancer.     

Targeted therapy against human lung cancer in nude mice by high-affinity recombinant antimesothelin single-chain Fv immunotoxin

Several tumors, including mesothelioma and ovarian cancer, can overexpress mesothelin, a glycosylphosphatidylinositol-linked differentiation glycoprotein. The membrane-bound type of mesothelin is found in the blood of cancer patients at a very low level, which makes mesothelin a good candidate for targeted therapy of certain cancers. An antimesothelin disulfide-linked Fv (SS1 Fv) was fused to a truncated mutant of Pseudomonas exotoxin A to produce the recombinant immunotoxin SS1(dsFv)-PE38, which has a high binding affinity to mesothelin (Kd = 0.7 nM). Our studies in vitro showed that SS1(dsFv)-PE38 is significantly more cytotoxic to the high-mesothelin-producing NCI-H226 human non-small cell lung cancer cells than to human lung adenocarcinoma PC14PE6 cells, which do not express mesothelin. When administered at a nontoxic dose of 500 microg/kg on days 7, 9, and 11 to nude mice injected i.v. with the two human lung cancer cell lines, SS1(dsFv)-PE38 selectively inhibited experimental lung metastases produced by the mesothelin-producing NCI-H226 cells. Our data indicate that mesothelin-producing squamous cell carcinoma of the lung may be a good target for this immunotoxin.     

肺癌患者血清特异性核酸适配体的筛选及鉴定

目的以磁珠为筛选介质,利用实时定量-PCR和消减SELEX技术从肺癌血清中筛选得到高特异性、强亲和力的肺癌血清核酸适配体。方法以磁珠为载体、肺癌病人血清为筛选的靶标分子,通过消减SELEX技术及实时定量PCR技术,从随机ssDNA文库中筛选出与肺癌血清特异性结合的aptamers,将得到的第9轮富集文库扩增为dsDNA,送上海生工直接对筛选得到的核酸适配体库进行高通量测序。结果经过9轮筛选得到4条与肺癌血清高特异性结合的aptamers,测序结果显示均为不同序列。结论验证筛选出的与肺癌血清结合的aptamer,特异性检测表明,筛选得到的肺癌血清核酸适配体与肺癌血清的结合解离常数均在纳摩尔级水平,其中Seq-2、Seq-3、Seq-6、Seq-19号核酸适配体能高特异性结合肺癌血清,与正常人血清不结合,再通过200例肺癌血清和200例正常人血清进一步鉴定4条肺癌血清核酸适配体检出肺癌的阳性率,阳性检出率达91%以上,为肺癌的早期诊断提供了新方法。     

Anti‐VEGF DNA‐based aptamers in cancer therapeutics and diagnostics

The vascular endothelial growth factor (VEGF) family and its receptors play fundamental roles not only in physiological but also in pathological angiogenesis, characteristic of cancer progression. Aiming at finding putative treatments for several malignancies, various small molecules, antibodies, or protein‐based drugs have been evaluated in vitro and in vivo as VEGF inhibitors, providing efficient agents approved for clinical use. Due to the high clinical importance of VEGF, also a great number of anti‐VEGF nucleic acid‐based aptamers—that is, oligonucleotides able to bind with high affinity and specificity a selected biological target—have been developed as promising agents in anticancer strategies. Notable research efforts have been made in optimization processes of the identified aptamers, searching for increased target affinity and/or bioactivity by exploring structural analogues of the lead compounds. This review is focused on recent studies devoted to the development of DNA‐based aptamers designed to target VEGF. Their therapeutic potential as well as their significance in the construction of highly selective biosensors is here discussed.     

Identification of a novel DNA aptamer that selectively targets lung cancer serum

Lung cancer is the leading cause of cancer-related deaths worldwide. Early diagnosis and treatment is critical to improving the 5 year survival rate of lung cancer. The identification of new options for early-stage diagnosis and therapy of lung cancer still represents a crucial challenge. Therefore, a new diagnostic method is urgently needed. In this study, we used a new modified SELEX, called serum-SELEX, to isolate aptamers that can specifically bind lung cancer serum, without any prior knowledge of their target. Among the obtained candidate aptamer sequences, Ap-LC-19 was identified as the optimal aptamer probe with the lowest dissociation constant (K_d) value of 15 ± 8.6 nM and higher affinity assessed by qPCR. Furthermore, this molecule could be a suitable aptamer for lung cancer serum and could be used as a recognition element in aptamer-based biosensors for efficient early diagnosis of lung cancer or as an innovative tool for targeted therapy. In addition, we performed MALDI-TOF MS followed by secondary peptide sequencing MS analysis for the identification of the aptamer targeted proteins. CLEC3B could be useful biomarkers for early detection of lung cancer and in monitoring its evolution.

Lung cancer is the leading cause of cancer-related deaths worldwide.     

Pancreatic cancer circulating tumor cells: applications for personalized oncology

Introduction: Pancreatic cancer (PC) is a highly lethal disease, in part because of early metastasis, late diagnosis, and limited treatment options. Circulating tumor cells (CTCs) are cancer cells that have achieved the metastatic step of intravasation, and are thus a unique source of biomarkers with potential applications in the staging, prognostication, and treatment of PC.

Areas covered: This review describes the use of CTCs in PC, including isolation methods, the significance of CTC enumeration, and studies examining phenotypic and molecular characteristics of CTCs. We also speculate on future directions for PC CTC research such as single-cell analysis and CTC culture.

Expert commentary: CTCs represent a potential unique serial source of cancer tissue via a convenient and minimally invasive blood draw. Recent development of isolation methods that allow for the release of viable CTCs with unaltered molecular characteristics has set the stage for single-cell analysis and ex vivo culture. Although there is significant potential for CTCs as a biomarker to impact PC from diagnosis to therapy, there still remain a number of challenges to the routine implementation of CTCs in the clinical management of PC.     

利用常见血清肿瘤标志物构建肺癌病理分型的风险预测模型

目的 联合运用6种常见血清肿瘤标志物建立预测肺癌常见病理分型的风险模型.方法 回顾性分析2012年5月至2013年5月新疆医科大学附属肿瘤医院收治的342例肺癌患者和91例肺部影像学可疑并确诊为非肺癌患者的临床资料.对6种肿瘤标志物进行热图分析,得到肿瘤标志物和肺癌各病理亚型的关系.Logistic回归分析筛选各病理亚...