Liquid biopsy in patients with hepatocellular carcinoma: Circulating tumor cells and cell-free nucleic acids

Hepatocellular carcinoma(HCC), with its high incidence and mortality rate, is one of the most common malignant tumors. Despite recent development of a diagnostic and treatment method, the prognosis of HCC remains poor. Therefore, to provide optimal treatment for each patient with HCC, more precise and effective biomarkers are urgently needed which could facilitate a more detailed individualized decision-making during HCC treatment, including the following; risk assessment, early cancer detection, prediction of treatment or prognostic outcome. In the blood of cancer patients, accumulating evidence about circulating tumor cells and cell-free nucleic acids has suggested their potent clinical utilities as novel biomarker. This concept, so-called "liquid biopsy" is widely known as an alternative approach to cancer tissue biopsy. This method might facilitate a more sensitive diagnosis and better decision-making by obtaining genetic and epigenetic aberrations that are closely associated with cancer initiation and progression. In this article, we review recent developments based on the available literature on both circulating tumor cells and cell-free nucleic acids in cancer patients, especially focusing on Hepatocellular carcinoma.

Liquid biopsy in patients with pancreatic cancer: Circulating tumor cells and cell-free nucleic acids

Despite recent advances in surgical techniques and perioperative management, the prognosis of pancreatic cancer(PCa) remains extremely poor. To provide optimal treatment for each patient with Pca, superior biomarkers are urgently needed in all phases of management from early detection to staging, treatment monitoring, and prognosis. In the blood of patients with cancer, circulating tumor cells(CTCs) and cell-free nucleic acids(cf NAs), such as DNA, m RNA, and noncoding RNA have been recognized. In the recent years, their presence in the blood has encouraged researchers to investigate their potential use as novel blood biomarkers, and numerous studies have demonstrated their potential clinical utility as a biomarker for certain types of cancer. This concept, called "liquid biopsy" has been focused on as a less invasive, alternative approach to cancer tissue biopsy for obtaining genetic and epigenetic aberrations that contribute to oncogenesis and cancer progression. In this article, we review the available literature on CTCs and cfN As in patients with cancer, particularly focusing on PCa, and discuss future perspectives in this field.

Liquid biopsy of gastric cancer patients:Circulating tumor cells and cell-free nucleic acids

To improve the clinical outcomes of cancer patients, early detection and accurate monitoring of diseases are necessary. Numerous genetic and epigenetic alterations contribute to oncogenesis and cancer progression, and analyses of these changes have been increasingly utilized for diagnostic, prognostic and therapeutic purposes in malignant diseases including gastric cancer (GC). Surgical and/or biopsy specimens are generally used to understand the tumor-associated alterations; however, those approaches cannot always be performed because of their invasive characteristics and may fail to reflect current tumor dynamics and drug sensitivities, which may change during the therapeutic process. Therefore, the importance of developing a non-invasive biomarker with the ability to monitor real-time tumor dynamics should be emphasized. This concept, so called “liquid biopsy”, would provide an ideal therapeutic strategy for an individual cancer patient and would facilitate the development of “tailor-made” cancer management programs. In the blood of cancer patients, the presence and potent utilities of circulating tumor cells (CTCs) and cell-free nucleic acids (cfNAs) such as DNA, mRNA and microRNA have been recognized, and their clinical relevance is attracting considerable attention. In this review, we discuss recent developments in this research field as well as the relevance and future perspectives of CTCs and cfNAs in cancer patients, especially focusing on GC.

Role of cell-free DNA for predicting incidence and outcome of patients with ischemic stroke

Early diagnosis and prognosis of ischemic stroke remains a critical challenge in clinical settings.A blood biomarker can be a promising quantitative tool to represent the clinical manifestations in ischemic stroke.Cell-free DNA(cfDNA)has recently turned out to be a popular circulating biomarker due to its potential relevance for diagnostic applications in a variety of disorders.Despite bright outlook of cfDNA in clinical applications,very less is known about its origin,composition,or function.Several recent studies have identified cell-derived mitochondrial components including mitochondrial DNA(mtDNA)in the extracellular spaces including blood and cerebrospinal fluid.However,the time course of alterations in plasma mtDNA concentrations in patients after an ischemic stroke is poorly understood.DNA is thought to be freed into the plasma shortly after the commencement of an ischemic stroke and then gradually decreased.However,the importance of cell-free mtDNA(cf-mtDNA)in ischemic stroke is still unknown.This review summarizes about the utility of biomarkers which has been standardized in clinical settings and role of cfDNA including cfmtDNA as a non-invasive potential biomarker of ischemic stroke.

PNA与引物的连接方法以及探讨潜在的价值

目的探讨一种基于点击化学反应将PNA和引物进行连接的新方法,挖掘PNA-引物具有选择性扩增cffDNA的潜力。方法采用点击化学将PNA与引物进行连接,通过变性聚丙烯酰胺凝胶电泳、高效液相色谱仪以及质谱分析对PNA-引物进行表征。最后,以DNA短片段和长片段分别模拟cffDNA和母源DNA,将包含相同摩尔质量的长短片段混合溶液通过聚合酶链式反应确定连接后的产物性能。结果PNA与引物成功连接,且连接物同时具备二者的特性。PNA-引物特异性富集DNA短片段的效率高达83.20%。结论本研究构建的新方法有望从高母源性DNA背景下,选择性地富集cffDNA,该方法同时具有通量高、成本低的特点,为cffDNA的分离及纯化提供新的思路。

孕妇外周血中胎儿游离DNA在产前诊断中的应用

孕妇外周血中胎儿游离DNA的发现,为无创性产前诊断开辟了一条新途径。检测孕妇血浆中特异的胎儿DNA序列已被用于胎儿性连锁疾病鉴定、RhD血型检查和多种单基因遗传病的产前诊断;游离DNA水平的变化还可被用作某些妊娠相关疾病如先兆子、早产和胎儿染色体疾病的临床诊断新指标。本文对母体外周血中胎儿游离DNA的生化特征及其临床应用研究,进行了总结和分析。

应用巢式PCR扩增孕妇血浆中胎儿SRY基因

目的:利用孕妇血浆中胎儿DNA进行无创产前诊断。方法:对包括10例X连锁隐性遗传病携带者的60例14～22孕周孕妇的血浆DNA进行巢式PCR,扩增胎儿来源的Y染色体特异性SRY基因。结果:38例妊娠男胎的孕妇中32例出现SRY基因扩增带,灵敏度为84.2%。22例妊娠女胎的孕妇中4例出现SRY扩增带,特异性为81.8%,总符合率为83.3%(50/60)。10例X连锁隐性遗传病携带者中7例妊娠男胎的孕妇中6例检出SRY基因,3例妊娠女胎的孕妇均未检出SRY基因。结论:采用巢式PCR技术检测母体血浆中的胎儿DNA进行产前性别鉴定具有较高的灵敏度和特异性,在遗传病的产前诊断中具有很大的应用前景。

基于母体血浆胎盘源及胚胎源核酸分子的早期妊娠诊断及早期胎儿性别鉴定技术

动物早期妊娠诊断及早期胎儿性别鉴定是繁殖技术中的关键技术。奶牛早期妊娠诊断可及时消除配种后假孕、妊娠后假发情等不利因素,胎儿性别早期鉴定可提高母牛犊的出生比例。近年来,随着孕妇血浆/血清中的胎儿源性游离DNA及胎盘源性游离RNA的发现,使人的早期性别鉴定技术及产前早期诊断技术的研究取得突破性进展;同时也使胎盘特异性表达基因的无创伤性研究成为可能。这些均为单胎动物,特别是奶牛的早期妊娠诊断及早期性别鉴定技术提供了理论依据及技术支持。

孕妇外周血中胎儿核酸研究进展

1997年,Lo等首次报道在母体循环中发现了游离胎儿DNA,为无创产前诊断开辟了新的领域。近年来,从孕妇外周血中直接提取胎儿DNA进行产前诊断日益受到重视。虽然孕妇血中胎儿核酸物质的来源目前仍不清楚,应用相对简单的分子生物学技术已经可以进行胎儿DNA的高灵敏度、高特异性的检测。这些方法已应用到胎儿RhD血型的诊断、胎儿性别鉴定、异常妊娠及染色体综合征的诊断等领域。研究表明胎儿核酸分析前某些因素的变化以及分析方法的不同都会对孕妇外周血中胎儿游离核酸分析产生影响,把握这些因素将使此方法在产前诊断中具有更广阔的应用前景。

子痫前期孕妇外周血游离DNA的研究进展

妊娠高血压疾病,尤其是子痫前期(PE),是世界范围内孕产妇和围产期发病率和病死率增加的主要原因之一。而孕妇血液胎儿游离的DNA(cff-DNA)可作为早期预测PE的发生发展。并且总结分析cff-DNA与PE的相关性及其可能存在的机制,进一步说明PE的发生可能与DNA甲基化相关,同时探讨其作为生物标志物的局限性,为有效预测子痫前期提供参考。

全球无创产前检测专利现状及发展趋势

本文基于全球无创产前检测(NIPT)相关专利文献,对全球专利申请态势、专利布局、创新主体以及中国专利申请法律状态进行分析,详细介绍中国专利技术分支。分析表明,中国NIPT领域与全球领先技术还有一定差距,但差距不是很大,且在相关政策支持下正在蓬勃发展。华大基因的申请数量和质量均占据一定优势,走在中国甚至世界前列。中国本土主流测序公司均具有一定的专利储备,新兴检测公司发展势头迅猛。与此同时,中国NIPT专利获得有效保护的专利占比不高,申请和保护策略有待提高。

Dimethylamino group modified polydopamine nanoparticles with positive charges to scavenge cell-free DNA for rheumatoid arthritis therapy

Excessive cell-free DNA(cfDNA)released by damaged or apoptotic cells can cause inflammation,impacting the progression of rheumatoid arthritis(RA).cfDNA scavengers,such as cationic nanoparticles(NPs),have been demonstrated as an efficient strategy for treating RA.However,most scavengers are limited by unfavorable biocompatibility and poor scavenging efficacy.Herein,by exploiting the favorable biocompatibility,biodegradability and bioadhesion of polydopamine(P),we modified P with dimethylamino groups to form altered charged DPs to bind negatively charged cfDNA for RA therapy.Results showed that DPs endowed with superior binding affinity of cfDNA and little cytotoxicity,which effectively inhibited lipopolysaccharide(LPS)stimulated inflammation in vitro,resulting in the relief of joint swelling,synovial hyperplasia and cartilage destruction in RA rats.Significantly,DPs with higher DS of bis dimethylamino group exhibited higher positive charge density and stronger cfDNA binding affinity,leading to excellent RA therapeutic effect among all of the treated groups,which was even close to normal rats.These finding provides a novel strategy for the treatment of cfDNA-associated diseases.

羊水悬液中游离胎儿核酸在产前诊断中的作用

传统的产前诊断方法是在妊娠中期通过羊膜腔穿刺获取羊水,从羊水中分离胎儿细胞进行培养,对胎儿的染色体异常进行诊断。近年研究证实在羊水悬液中存在大量游离胎儿核酸,可利用从羊水悬液中提取的游离胎儿DNA和RNA,对胎儿的染色体异常和胎儿发育状况进行诊断和评估。这些发现使利用废弃的羊水悬液获取胎儿遗传物质进行产前诊断成为可能,为研究胎儿发育和病理状态开辟了一条新的途径。作者现将近年的相关研究进行综述如下。

异常血红蛋白合并遗传性持续性胎儿血红蛋白增多症患者一例的基因分析

目的 对1例异常血红蛋白合并遗传性持续性胎儿血红蛋白增多症（HPFH）患者进行基因分析.方法 患者男,26岁,2010年9月到广州金域医学检验中心进行体检,经血液学分析、血红蛋白电泳、红细胞渗透脆性分析后,疑为异常血红蛋白合并HPFH病患者.应用常规血液学检查、碱性琼脂糖凝胶血红蛋白电泳对先证者进行表型分析；通过对HBB基因直接测序检测异常血红蛋白的突变类型；应用多重连接酶依赖探针扩增技术（MLPA）检测整个β-珠蛋白基因簇有无缺失及缺失的大致范围；应用跨越断裂点聚合酶链反应（gap-PCR）进行跨越断裂点PCR扩增,对此扩增产物进行基因测序分析,并将其结果与参考序列NC_000011.9进行比对,判断具体的断裂位点.结果 通过对HBB基因进行直接测序,发现该异常血红蛋白为Ta-Li（ HBB：c.250G＞T）,通过MLPA结合gap-PCR以及对产物进行基因测序,发现患者存在β-珠蛋白基因的大片段缺失,缺失范围为NC_000011.9：g.5222878_5250288del,即患者的基因型为异常血红蛋白Ta-Li合并东南亚型遗传性持续性胎儿血红蛋白增多症（SEA-HPFH）.结论 联合应用MLPA、gap-PCR和基因测序技术可能有助于明确先证者的基因型.

母血浆中胎儿游离核酸与无创性产前亲子鉴定

近年来,涉及胎儿亲权鉴定的案例逐渐增多。传统胎儿产前亲权鉴定常需有创性取样,故存在一定的风险。母血浆中胎儿游离核酸的发现,给无创性产前亲权鉴定带来了新的契机。本文主要就母血浆中胎儿游离核酸及其在无创性产前亲权鉴定中的应用价值和前景进行简要介绍。

孕妇外周血中胎儿核酸的研究新进展

从孕妇外周血中直接提取胎儿成分进行产前诊断日益受到重视。明确胎儿细胞或者核酸在母血中的存在形式、正常含量以及病理情况下的改变,对非侵入性的产前诊断具有很重要的临床意义,同时孕妇外周血胎儿核酸在病理性产科及优生优育等方面都有了新的广泛的研究。

Circulating Tumor DNA as Biomarkers for CancerDetection

Detection of circulating tumor DNAs(ct DNAs) in cancer patients is an important component of cancer precision medicine ct DNAs. Compared to the traditional physical and biochemical methods, blood-based ct DNA detection offers a non-invasive and easily accessible way for cancer diagnosis, prognostic determination, and guidance for treatment. While studies on this topic are currently underway, clinical translation of ct DNA detection in various types of cancers has been attracting much attention, due to the great potential of ct DNA as blood-based biomarkers for early diagnosis and treatment of cancers. ct DNAs are detected and tracked primarily based on tumorrelated genetic and epigenetic alterations. In this article, we reviewed the available studies on ct DNA detection and described the representative methods. We also discussed the current understanding of ct DNAs in cancer patients and their availability as potential biomarkers for clinical purposes. Considering the progress made and challenges involved in accurate detection of specific cell-free nucleic acids, ct DNAs hold promise to serve as biomarkers for cancer patients, and further validation is needed prior to their broad clinical use.