肿瘤循环DNA的研究进展

肿瘤患者循环DNA不仅在数量上明显高于健康人, 而且还可以出现与肿瘤DNA相同的性质改变。肿瘤循环DNA的特征性变化在肿瘤的早期诊断、疗效评价以及肿瘤随访观察等方面有一定临床意义。本文就循环DNA的来源、释放机制、基因修饰变异、水平变化、完整性, 以及循环DNA的研究方法和局限性等方面进行综述。     

血中循环DNA启动子甲基化与妇科肿瘤的关系

妇科恶性肿瘤患者外周血中存在着循环DNA,其含量明显高于健康人水平且具有与肿瘤原发灶相一致的基因异常改变,DNA基因启动子甲基化是基因异常中一项重要的表观遗传学改变,是导致肿瘤抑癌基因、DNA错配修复基因、转移抑制基因等表达沉默的重要分子生物学基础.目前已检测到妇科恶性肿瘤患者外周血循环DNA中有多种基因存在异常甲基化,且与肿瘤患者的临床病理特征存在不同程度的相关性.这使得非创伤性早期诊断肿瘤、及时发现肿瘤的复发或转移、监测疗效、预测预后成为可能.     

肝癌患者肿瘤循环DNA的检测

一、肿瘤循环DNA的发展简史早在50多年前,也就是Watson和Crick阐明DNA双螺旋结构前五年,Mandel和Me'tais在人体血浆中发现了核酸[1].1975年,Leon[2]发现肿瘤患者比正常健康人血液中有更多的循环DNA.随着PCR方法在循环DNA领域的应用,人们在胰腺癌及黑色素淋巴瘤患者血浆DNA中,首次检测到N-ras及K-ras基因突变[3,4].     

肿瘤患者血清DNA甲基化的研究进展

血清DNA是循环核酸的一种，DNA甲基化与肿瘤的发生发展有密切关系；甲基化特异性PCR(MSP)是血清DNA甲基化检测的灵敏方法，血清DNA甲基化在肿瘤的早期诊断、分期、治疗及预后监测等方面有重要意义，是目前肿瘤分子生物学研究热点之一，具有极大的应用价值。     

循环DNA、循环肿瘤细胞端粒酶与肿瘤早期诊断

肿瘤患者循环DNA水平的增高、肿瘤特征性基因改变及循环肿瘤细胞端粒酶活性的表达,不仅可出现在肿瘤的晚期,也可发生在肿瘤的早期阶段。循环DNA和肿瘤细胞端粒酶的联合检测,对于肿瘤早期诊断有重要意义。     

循环肿瘤DNA在结直肠癌临床诊治中应用的研究进展

结直肠癌发病率在我国的恶性肿瘤中占第 3 位,且有上升趋势,发病率高达 37/10 万人,其中约 60%的患者确诊时已是中晚期[1]. 目前临床上结直肠癌的诊疗难点包括:难以早发现;肿瘤标志物灵敏度低、特异性差;组织活检不易操作;诊疗效果难以及时监测;肿瘤的异质性导致个体化用药指导和耐药性监测手段有限. 血浆游离循环肿瘤DNA(circulating tumor DNA,ctDNA)[2]是由肿瘤细胞释放到血浆中的单链或者双链 DNA 片段, 携带有与原发肿瘤组织相一致的分子遗传学信息. 它是来自肿瘤细胞的体细胞突变,与遗传突变不同,遗传突变存在于体内每个细胞,而ctDNA 可在外周循环中直接检测. ctDNA 检测作为一种无创的检测方法,能够真实地反映实体瘤组织中的基因突变图谱与频率,可以作为一种治疗效果评估及治疗后临床随访的重要监测指标, 尤其是对于一些不具有典型临床症状、检查无特异性和诊断困难的肿瘤可避免复杂的、具有创伤性的活检. 因此,作为一种新的肿瘤标志物,ctDNA 将在肿瘤的诊断、 治疗及预后检测等方面发挥重要的作用.本文对循环肿瘤 DNA 在结直肠癌患者临床诊治中应用的最新进展进行综述.     

血循环核酸作为肿瘤诊断生物标志物的研究进展

循环核酸(circulating nucleic acids,CNAs)是一种存在于动植物和人体液中的细胞外游离状态核酸,包括循环DNA、循环RNA以及新发现的微小RNA(miRNA).血液不同于其他体液,分布于全身各处,因此,血CNAs具有良好的临床应用前景.由于生理状况、疾病种类和病程的不同,CNAs在血清和血浆中存在的种类和数量也会发生变化.本文中,我们主要介绍了血CNAs的发现过程、分子结构、生物学特性、检测方法以及与肿瘤发生、发展关系等方面的研究进展.一、肿瘤患者血CNAs的发现1948年,法国科学家首次报道,在人血浆中可分离得到游离形式的核酸.由于当时人们认为,核酸需在完整细胞内传递生物遗传信息,因此,这一发现并未引起广泛关注.数十年后,植物学家Anker和Stroun发现,植物冠瘿肿瘤的转移是通过游离核酸来完成的.随后,人们证实,肿瘤患者血液中存在游离形式的DNA,血液中循环DNA不仅发生基因突变,还包括了微卫星改变、倒位、缺失和甲基化等.至此,CNAs在肿瘤诊断方面的研究有了快速发展.     

循环肿瘤细胞临床应用的新进展

循环肿瘤细胞(Circulating tumor cells,CTCs)是指释放到血液中的罕见肿瘤细胞,被认为是肿瘤血液转移的关键步骤之一.循环肿瘤细胞的检测对于肿瘤的诊断、治疗和预后判定都具有非常重要的意义,是目前肿瘤研究的热点之一.本文将从预测生物标志物、治疗反应、预后、血浆肿瘤DNA四个方面介绍循环肿瘤细胞临床应用的最新研究进展.     

液体活检-通向精准医疗的桥梁

组织活检是癌症的标准诊断程序,然而,由于肿瘤异质性和进化,基于组织活检的癌症诊断在癌症发展、预后的评估中具有局限性.液体活检是检测患者体液中含原发肿瘤生物特性分子的技术,包括循环肿瘤细胞、循环肿瘤DNA和外泌体等的检测.液体活检被广泛的应用于辅助诊断、疗效评价、预测预后、监测复发转移、辅助靶向治疗等方面.本文将对液体活检的生物特性及临床应用进展进行综述.     

液体活检在食管鳞状细胞癌中的应用及展望

食管癌是世界上最常见的恶性肿瘤之一,常见的病理类型包括鳞状细胞癌和腺癌,其中鳞状细胞癌占食管癌的90％.食管鳞状细胞癌(ESCC)发病率高,早期诊断困难,复发率高,预后欠佳,目前仍缺乏有效的检测方法.液体活检作为一种新兴技术,包括对循环肿瘤细胞(CTC)、循环肿瘤DNA(ctDNA)、循环肿瘤RNA(ctRNA)、肿瘤来源的外泌体、肿瘤相关血小板(TEP)及循环肿瘤相关微粒等进行检测,可在各类肿瘤的早期诊断、治疗及预后判断中发挥重要作用.本文围绕液体活检技术在ESCC中的最新研究进展作一综述,旨在探讨循环肿瘤标志物在ESCC诊断、治疗及预后判断中的作用及局限性.     

Analysis of tumor-derived DNA in plasma and bone marrow fluid in lung cancer patients

Liquid biopsies such as circulating tumor DNA in plasma and disseminated tumor cells in the bone marrow are currently available. However, it is unclear which types of samples are appropriate for detecting tumor DNA in these biopsies. Here, we collected primary tumors, pulmonary venous blood, peripheral blood, and rib bone marrow fluid from 10 lung cancer patients. Targeted deep sequencing was performed to identify mutations across 70 specimens. As a result, a total of 43 mutations were identified in the primary tumors. The mutation in the tumors was also identified in circulating tumor DNA in the pulmonary venous and peripheral blood in two patients. These patients showed poor prognosis, as compared to the other patients. However, no mutation was identified in the bone marrow in any of the patients. These results demonstrated that circulating tumor DNA in plasma is more sensitive and clinically useful as a biomarker as compared to DNA in bone marrow fluid.     

Characterization of cell-free circulating DNA in plasma in patients with prostate cancer

Cell-free circulating DNA in plasma and serum may serve as a biomarker for malignant tumor detection and follow up in patients with a variety of solid tumors including prostate cancer. In healthy patients, DNA is normally released from an apoptotic source which generates small fragments of cell-free DNA, whereas cancer patients have cell-free circulating DNA that originated from necrosis, autophagy, or mitotic catastrophe. Cell-free circulating DNA levels were measured by a quantitative real-time PCR method with a set of primers targeted to amplify the consensus ALU apoptotic versus necrotic origin. Prostate cancer patients before and 3 months after diagnosis showed cell-free circulating DNA released at apoptotic and non-apoptotic cell death. Interestingly, all patients after 6 months demonstrated DNA released at non-apoptotic cell. The principal source of cell-free circulating DNA is of apoptotic and non-apoptotic cell death. However, during treatment, this feature could change. Therefore, the study of cell-free circulating DNA would be important to follow the evolution of the disease during the treatment.     

Monitor tumor burden with circulating tumor DNA

There is a need to identify better biomarkers to monitor diseases and/or assess therapeutic responses. For those with cancer, one can identify DNA fragments that contain somatic mutations originating in the tumor DNA in plasma or serum. There have been several early studies suggesting that advances in sequencing technologies will allow identification of somatic genomic alterations that can be used to monitor tumor dynamics. Dawson et al. investigated circulating cell-free DNA carrying tumor specific alterations in patients with breast cancer. The authors compared CT imaging from 30 women with metastatic breast cancer receiving treatment, using two assays for circulating tumor DNA, CA 15-3, and CTCs. Taken the two methods together circulating tumor DNA was detected in 29 or 30 women (97%) and 115 of 141 plasma samples (82%). Circulating tumor DNA levels showed a greater dynamic range and greater correlation with changes in tumor burden than did CA 15-3 or CTC. The relatively small study showed that circulating tumor DNA has a superior sensitivity to other circulating biomarkers and a dynamic range that correlates with tumor burden.     

非小细胞肺癌EGFR突变的分子影像学在体检测

靶向药物表皮生长因子受体酪氨酸激酶抑制剂(epidermal growth factor receptor tyrosine kinase inhibitor,EGFR-TKI)改变了非小细胞肺癌的治疗格局,研究表明只有EGFR敏感突变人群能从中获益.EGFR突变检测的主流方法是针对EGFR的DNA序列进行分析,标本可以是手术或穿刺获取的肺癌组织、胸水肿瘤细胞、循环肿瘤细胞、外周血游离DNA,其最大的缺点是无法分析EGFR突变的异质性.针对EGFR在蛋白质水平进行突变检测分析的技术尚不成熟,但随着分子影像学的发展,基于正电子发射型计算机断层显像(positron emission computed tomography,PET)-计算机断层扫描(computed tomography,CT)的靶向EGFR分子探针的研发,使得在体检测肺癌组织的EGFR突变状态成为了可能,而且可以检测EGFR突变的异质性.本文综述了目前靶向EGFR突变的分子探针的研究结果及进展.     

The feasibility of using mutation detection in ctDNA to assess tumor dynamics

For many decades it has been known that tumor DNA is shed into the blood. As a consequence of technological limitations, researchers were unable to comprehensively characterize circulating DNA. The advent of ultrasensitive and highly specific molecular assays has provided a comprehensive profile of the molecular characteristics and dynamics of circulating DNA in healthy subjects and cancer patients. With these new tools in hand, significant interest has been provoked for an innovative type of tumor biopsy termed a “liquid biopsy”. Liquid biopsies are obtained by minimal invasive blood draws from cancer patients. Circulating cancer cells, exosomes and a variety of molecules contained within the liquid biopsy including cell‐free circulating tumor DNA (ctDNA) can serve as promising tools to track cancer evolution. Attractive features of ctDNA are that ctDNA isolation is straightforward, ctDNA levels increase or decrease in response to the degree of tumor burden and ctDNA contains DNA mutations found in both primary and metastatic lesions. Consequently, the analysis of circulating DNA for cancer‐specific mutations might prove to be a valuable tool for cancer detection. Moreover, the capacity to screen for ctDNA in serial liquid biopsies offers the possibility to monitor tumor progression and responses to therapy and to influence treatment decisions that ultimately may improve patient survival. Here we focus on mutation detection in ctDNA and provide an overview of the characteristics of ctDNA, detection methods for ctDNA and the feasibility of ctDNA to monitor tumor dynamics. Current challenges associate with ctDNA will also be discussed.     

DETECTION OF TUMOR MUTANT DNA IN PLASMA OF PATIENT WITH COLORECTAL CANCER

Colorectal cancer accounts for more than 90% of malignancies of the large bowel and is the second most common cause of cancer mortality in the adult American population[1]. Also the incidence of colorectal cancer increases by 4.2% per year in China[2]. Approximately 40% of affected individuals will ultimately die from the cancer. Early diagnosis and prompt treatment are mandatory to prevent this devastating disease. Thus, the search for markers which might provide reliable information for ear…     

DNA fragments in the blood plasma of cancer patients: quantitations and evidence for their origin from apoptotic and necrotic cells

Increased levels of DNA fragments have frequently been found in the blood plasma of cancer patients. Published data suggest that only a fraction of the DNA in blood plasma is derived from cancer cells. However, it is not known how much of the circulating DNA is from cancer or from noncancer cells. By quantitative methylation-specific PCR of the promoter region of the CDKN2A tumor suppressor gene, we were able to quantify the fraction of plasma DNA derived from tumor cells. In the plasma samples of 30 unselected cancer patients, we detected quantities of tumor DNA from only 3% to as much as 93% of total circulating DNA. We investigated possible origins of nontumor DNA in the plasma and demonstrate here a contribution of T-cell DNA in a few cases only. To investigate the possibility that plasma DNA originates from apoptotic or necrotic cells, we performed studies with apoptotic (staurosporine) and necrotic (staurosporine plus oligomycin) cells in vitro and with mice after induction of apoptotic (anti-CD95) or necrotic (acetaminophen) liver injury. Increasing amounts of DNA were found to be released in the supernatants of cells and in the blood plasma samples of treated animals. A clear discrimination of apoptotic and necrotic plasma DNA was possible by gel electrophoresis. The same characteristic patterns of DNA fragments could be identified in plasma derived from different cancer patients. The data are consistent with the possibility that apoptotic and necrotic cells are a major source for plasma DNA in cancer patients.