新的肿瘤分子标记物--肿瘤相关循环DNA

肿瘤患者外周血循环DNA的存在及相关改变为肿瘤的诊断和监测提供了新的靶分子,血浆／血清作为容易获得的检测对象比传统的癌组织活检具有更大的使用弹性和临床应用价值,循环DNA作为新的肿瘤标志物的研究在不久的未来将成为科学研究的热点。     

DNA 甲基化及其与胃癌相关研究进展

DNA甲基化是表观遗传学中的研究热点,与肿瘤的发生、发展、诊断、治疗、预后等相关。胃癌的发生、发展与DNA甲基化状态改变关系密切,研究胃癌相关基因DNA甲基化状态的改变有助于胃癌的早期发现、诊断、治疗及预后。因此,研究胃癌相关基因的甲基化状态具有一定的临床价值。     

DNA甲基化及其与胃癌相关研究进展

DNA甲基化是表观遗传学中的研究热点,与肿瘤的发生、发展、诊断、治疗、预后等相关。胃癌的发生、发展与DNA甲基化状态改变关系密切,研究胃癌相关基因DNA甲基化状态的改变有助于胃癌的早期发现、诊断、治疗及预后。因此,研究胃癌相关基因的甲基化状态具有一定的临床价值。     

晚期胃肠道肿瘤患者血浆游离DNA片段的检测、定量

目的探讨肿瘤患者循环血液中DNA改变,及对提高肿瘤患者治愈率和生存率的临床实际意义。方法随机采集了2004年10月~12月大连医科大学附属二院经病理确诊的初治晚期胃、大肠癌患者的血标本18份,门诊健康体检者血标本16份,通过酚/氯仿提取法定量检测两组的血浆DNA片段,进行对照比较,分析健康人与晚期胃肠道肿瘤患者血浆游离DNA片段水平的差异、晚期胃肠道肿瘤患者血浆游离DNA片段水平与预后相关指标的相关性。结果晚期胃肠道肿瘤组血浆游离DNA片段水平高于正常健康对照组,其血浆游离DNA片段的均值分别为(346.3±81.62)μg/m l与(27.6±6.91)μg/m l,差异有显著性;胃癌、大肠癌患者两组间的血浆游离DNA片段差异无显著性,均值分别为:(350.8±110.92)μg/m l与(357.1±126.26)μg/m l;在预后相关指标的各因素中,晚期胃肠道肿瘤患者的血浆游离DNA片段水平与免疫状态、是否存在远处转移有明显相关性,其中与免疫状态呈负相关,与远处转移呈正相关。结论晚期胃肠道肿瘤患者血中的游离DNA片段水平较健康人高,可能与患者自身状态、病理分型、疾病状态有关;游离DNA片段可以作为检测晚期胃肠道肿瘤的特异性指标,还可以替代免疫状态及是否存在远处转移等指标评价远期预后,并有取材方便、检测时间短、可以定量分析的优点,对临床有一定的指导意义和应用价值。     

DNA甲基化与食管腺癌关系研究进展

癌症本质上是一种多种因素导致的基因疾病。作为肿瘤形成假说中的重要补充内容,表观遗传学已经成为新的研究中心。DNA甲基化是人类基因组发生最为常见的一种表观遗传学事件,因而研究甲基化与肿瘤的关系成为当前分子生物学的热点之一。这篇综述是关于DNA甲基化与食管腺癌的研究进展,包括DNA高甲基化异常与食管腺癌的发生,以及针对甲基化的检测手段,诊断,治疗以及预后。     

抑癌基因ARID1A在肿瘤中的研究进展

染色质重塑复合物相关基因在癌症中频繁突变,这种现象逐渐引起研究者的重视。然而,染色质重塑活动如何引起癌症发生,对此机理研究甚少。ARID1A是SWl／SNF（BRG1相关因子）染色质重塑复合物中的一个亚基,具有DNA结合活性,可以与富含AT的DNA序列特异性结合。近来基因组测序发现,ARID1A在卵巢癌、肝癌、胃癌、乳腺癌等肿瘤中频繁发生突变,这些突变导致ARID1A在肿瘤中表达降低,表明ARID1A是个潜在的抑癌基因。该文将针对ARID1A在各种癌症中的缺失及失活机制、ARID1A的生物学功能和潜在抑癌机理以及与,临床预后之间关系等方面做一综述,以期为肿瘤诊断、治疗提供新思路。     

血浆游离DNA的临床应用新进展

血浆游离DNA(Plasma cell-free DNA,cf-DNA)游离于细胞外的机体内源性DNA,cf-DNA广泛存在于血液、脑脊液、滑膜液、支气管肺泡灌洗液和羊水等体液中。多数血浆DNA与蛋白质结合成为复合物,仅有少部分为游离的片段,受实验方法灵敏性和特异性的限制,cf-DNA的研究进展缓慢。随着分子检测技术的发展,虽然在健康人群的血浆中也可测量到一定量的cf-DNA,但是cf-DNA被发现在肿瘤、自身免疫性疾病、创伤和炎性反应等病理过程中有改变。目前cf-DNA在肿瘤的诊断和预后分析、产前诊断、感染性疾病等中均有应用价值。     

非小细胞肺癌患者血中DNA片段的检测、定量及临床意义的研究

目的探讨非小细胞肺癌患者血中游离DNA片段在临床上早期诊断、分期、预后方面的意义。方法随机采集2004年10月至12月大连医科大学附属二院经病理确诊的初治Ⅲ、Ⅳ期非小细胞肺癌患者的血标本15份,门诊健康体检者血标本15份,通过酚/氯仿提取法定量检测两组的血浆DNA片段,进行对照比较,分析非小细胞肺癌组内各因素(包括与诊断相关的肿瘤标记物,与预后相关的年龄、性别、一般状况及病理类型、疾病分期、免疫指标、是否转移等)与血浆游离DNA片段水平的相关性。结果非小细胞肺癌组血浆游离DNA片段水平高于正常健康对照组,其血浆游离DNA片段的均值分别为:(384.8±99.36)μg/ml与(13.9±3.60)μg/ml,差异有显著性,Ⅲ期与Ⅳ期非小细胞肺癌患者的血浆游离DNA片段差异有显著性,均值分别为:(290.4±91.82)μg/ml与(481.2±196.44)μg/ml,且非小细胞肺癌患者的血浆游离DNA片段与肿瘤标记物CA199、CYFRA21-1、ALP呈正相关性;在预后因素分析中,显示非小细胞肺癌患者的血浆游离DNA片段水平与一般状态、免疫状态、病理类型及是否存在远处转移等因素有明显相关性,其中与一般状态、免疫状态呈负相关,与远处转移呈正相关,与性别、年龄、治疗疗效等因素无相关性。结论非小细胞肺癌患者血中的游离DNA片段水平较健康人高,且不同病期有一定差异,可能与患者自身状态、病理分型、疾病状态有关;游离DNA片段可以作为检测非小细胞肺癌的特异性指标,还可以替代一般状态、免疫状态、病理类型、是否存在远处转移等指标评价远期预后,并有取材方便、检测时间短、可以定量分析的优点,对临床有一定的指导意义和应用价值。     

p53在乳腺癌中的作用

乳腺癌是一种世界范围内女性发病率较高的肿瘤之一,至今为止对其的诊断、病理机制、治疗及预后已有一定的了解.一部分科学家的对乳腺癌的研究主要集中在p53在其发病机理、治疗、预后中的作用的探讨.p53是目前被广泛关注的肿瘤抑制基因,它所编码的转录蛋白因子主要调节一系列与细胞周期抑制、衰老、凋亡、DNA的修复、新血管生成的反应相关的基因.主要就近几年对乳腺癌的研究中,p53基因在乳腺癌中的作用机制的进一步研究,p53做为分子标记在诊断和预后的意义,它的突变和等位基因的性质,以及以p53为基础的各种治疗手段中其与药物及其他基因最新的作用机制进行初步的阐述.     

胞外DNA的研究进展

胞外DNA(extracellular occurring DNA,eoDNA)是一种独立于细胞外的DNA,广泛存在于体液中。研究发现,eoDNA的浓度水平及其特异基因的改变能很好地反映疾病的发生和发展。随着生物技术的发展,eoDNA易获得、微创伤、预测早等优点引起了许多学者的关注,使得eoDNA成为非入侵疾病检测生物标记中的一颗新星。相对于健康人,肿瘤患者体内eoDNA浓度明显升高,这一特征已被研究者广泛验证,同时,研究还发现肿瘤患者eoDNA部分起源于肿瘤组织细胞,且这些DNA与肿瘤组织基因组有着相似的分子特征。这些研究成果为eoDNA取代肿瘤组织早期微创诊断肿瘤发生提供了理论基础。此外,在产检方面,从母体血浆中获取胎儿eoDNA并用以观察胎儿健康情况也日益得到学者关注。该文从eoDNA研究背景出发就其作为肿瘤诊断、预后以及产检生物标记的可能性及应用作一简单综述,并展望了eoDNA在临床疾病诊断的应用前景。     

DNA methylation signatures in circulating cell-free DNA as biomarkers for the early detection of cancer

Detecting cell-free DNA(cfDNA) or circulating tumor DNA(ctDNA) in plasma or serum could serve as a "liquid biopsy", which would be useful for numerous diagnostic applications. cfDNA methylation detection is one of the most promising approaches for cancer risk assessment. Here, we reviewed the literature related to the use of serum or plasma circulating cell-free DNA for cancer diagnosis in the early stage and their power as future biomarkers.     

Evaluation of Exome Sequencing to Estimate Tumor Burden in Plasma

Accurate estimation of systemic tumor load from the blood of cancer patients has enormous potential. One avenue is to measure the presence of cell-free circulating tumor DNA in plasma. Various approaches have been investigated, predominantly covering hotspot mutations or customized, patient-specific assays. Therefore, we investigated the utility of using exome sequencing to monitor circulating tumor DNA levels through the detection of single nucleotide variants in plasma. Two technologies, claiming to offer efficient library preparation from nanogram levels of DNA, were evaluated. This allowed us to estimate the proportion of starting molecules measurable by sequence capture (<5%). As cell-free DNA is highly fragmented, we designed and provide software for efficient identification of PCR duplicates in single-end libraries with a varying size distribution. On average, this improved sequence coverage by 38% in comparison to standard tools. By exploiting the redundant information in PCR-duplicates the background noise was reduced to ∼1/35000. By applying our optimized analysis pipeline to a simulation analysis, we determined the current sensitivity limit to ∼1/2400, starting with 30 ng of cell-free DNA. Subsequently, circulating tumor DNA levels were assessed in seven breast- and one prostate cancer patient. One patient carried detectable levels of circulating tumor DNA, as verified by break-point specific PCR. These results demonstrate exome sequencing on cell-free DNA to be a powerful tool for disease monitoring of metastatic cancers. To enable a broad implementation in the diagnostic settings, the efficiency limitations of sequence capture and the inherent noise levels of the Illumina sequencing technology must be further improved.     

循环microRNAs的外泌形成机制及其对乳腺癌侵袭和转移的影响

目前乳腺癌的临床诊疗主要依赖影像学和相对较少的预后/预测指标(如雌激素受体、孕激素受体、HER2等).这些生物标志物主要是基于原发肿瘤病灶的生物学检测,可用于转移或复发的检测指标很少,尤其是在切除肿瘤原发灶后,复发监测很困难.循环cell-free microRNAs(circulating cf-miRNAs,或简称circulating miRNAs)的发现为改变现有乳腺癌临床诊疗模式提供了可能.Cell-free miRNA通过外泌体、微囊或转运蛋白的主动外泌机制,可能在循环miRNA的形成中起着重要作用.Cell-free miRNA特别是circulating miRNA不仅自身可以作为信号分子影响肿瘤细胞和组织微环境,而且还可以与其他信号通路发生交互通讯来调控肿瘤部位新生血管的形成和肿瘤细胞表型的上皮-间质转换,影响乳腺癌的侵袭和转移.本文综述了循环miRNA的特征与分泌机制,特别是乳腺癌相关的循环miRNA参与作为一种液体活检生物标志物在乳腺癌诊断、预后评价和疗效评估的临床意义.     

Blood-based DNA methylation signatures in cancer: A systematic review

DNA methylation profiles are in dynamic equilibrium via the initiation of methylation, maintenance of methylation and demethylation, which control gene expression and chromosome stability. Changes in DNA methylation patterns play important roles in carcinogenesis and primarily manifests as hypomethylation of the entire genome and the hypermethylation of individual loci. These changes may be reflected in blood-based DNA, which provides a non-invasive means for cancer monitoring. Previous blood-based DNA detection objects primarily included circulating tumor DNA/cell-free DNA (ctDNA/cfDNA), circulating tumor cells (CTCs) and exosomes. Researchers gradually found that methylation changes in peripheral blood mononuclear cells (PBMCs) also reflected the presence of tumors. Blood-based DNA methylation is widely used in early diagnosis, prognosis prediction, dynamic monitoring after treatment and other fields of clinical research on cancer. The reversible methylation of genes also makes them important therapeutic targets. The present paper summarizes the changes in DNA methylation in cancer based on existing research and focuses on the characteristics of the detection objects of blood-based DNA, including ctDNA/cfDNA, CTCs, exosomes and PBMCs, and their application in clinical research.     

Detection of circulating tumor-derived material in peripheral blood of pediatric sarcoma patients: A systematic review

BackgroundDetection of circulating tumor-derived material (cTM) in the peripheral blood (PB) of cancer patients has been shown to be useful in early diagnosis, prediction of prognosis, and disease monitoring. However, it has not yet been thoroughly evaluated for pediatric sarcoma patients.MethodsWe searched the PubMed and EMBASE databases for studies reporting the detection of circulating tumor cells, circulating tumor DNA, and circulating RNA in PB of pediatric sarcoma patients. Data on performance in identifying cTM and its applicability in diagnosis, and evaluation of tumor characteristics, prognostic factors, and treatment response was extracted from publications.ResultsA total of 79 studies were assigned for the present systematic review, including detection of circulating tumor cells (116 patients), circulating tumor DNA (716 patients), and circulating RNA (2887 patients). Circulating tumor cells were detected in 76% of patients. Circulating DNA was detected in 63% by targeted NGS, 66% by shallow WGS, and 79% by digital droplet PCR. Circulating RNA was detected in 37% of patients.ConclusionOf the cTM from Ewing's sarcoma and rhabdomyosarcoma ctDNA proved to be the best target for clinical application including diagnosis, tumor characterization, prognosis, and monitoring of disease progression and treatment response. For osteosarcoma the most promising targets are copy number alterations or patient specific micro RNAs, however, further investigations are needed to obtain consensus on clinical utility.     

外周血循环RNA、微小RNA与肿瘤诊断*

循环核酸(circulating nucleic acid, CNA)是指存在于血液(血清或血浆)等体液中的细胞外游离DNA和RNA,与生理和病理状态下的细胞代谢密切相关,循环DNA和RNA水平的检测及其在基因诊断等方面的应用对于恶性肿瘤等疾病的诊断和监测具有十分重要的意义。近期人们在进行循环DNA、RNA分子检测的同时,在循环微小RNA(microRNA, miRNA)方面也做了许多的工作,研究结果使得与肿瘤相关的核酸分子检测这一领域变的更加让人振奋。在血液中检出肿瘤特异性的miRNA并将其作为肿瘤的生物学标志对于早期诊断肿瘤具有至关重要的意义。     

Elevated levels of cell-free circulating DNA in patients with acute dengue virus infection

Background

Apoptosis is thought to play a role in the pathogenesis of severe dengue and the release of cell-free DNA into the circulatory system in several medical conditions. Therefore, we investigated circulating DNA as a potential biomarker for severe dengue.

Methods and Findings

A direct fluorometric degradation assay using PicoGreen was performed to quantify cell-free DNA from patient plasma. Circulating DNA levels were significantly higher in patients with dengue virus infection than with other febrile illnesses and healthy controls. Remarkably, the increase of DNA levels correlated with the severity of dengue. Additionally, multivariate logistic regression analysis showed that circulating DNA levels independently correlated with dengue shock syndrome.

Conclusions

Circulating DNA levels were increased in dengue patients and correlated with dengue severity. Additional studies are required to show the benefits of this biomarker in early dengue diagnosis and for the prognosis of shock complication.     

Levels of circulating cell-free serum DNA in benign and malignant breast lesions

PURPOSES OF THE STUDY: We analyzed circulating cell-free DNA in the serum of patients with benign and malignant breast disease and in healthy individuals to determine its diagnostic value. BASIC PROCEDURES: Serum samples were obtained from 50 healthy individuals, 33 patients with malignant breast disease and 32 patients with benign breast disease. Circulatory DNA was extracted from serum samples. Cell-free DNA was quantified by real-time quantitative PCR for the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. Tissue samples from patients with malignant and benign breast lesions were histopathologically examined. MAIN FINDINGS: The mean levels of circulating cell-free DNA in serum samples were 41,149 genome equivalents (GE)/mL in patients with malignant disease, 30,826 GE/mL in patients with benign disease, and 13,267 GE/mL in healthy individuals. Healthy individuals had significantly lower levels of cell-free DNA than patients with malignant or benign breast disease (p=0.001, p=0.031). No significant difference was observed between malignant and benign disease. There was a correlation between cell-free DNA levels and tumor size but not with other tumor characteristics. PRINCIPAL CONCLUSION: Our results suggest that levels of circulating cell-free DNA in serum could have diagnostic value to discriminate between healthy individuals and patients with breast lesions but not between patients with malignant and benign breast lesions.     

Methylated DNA/RNA in Body Fluids as Biomarkers for Lung Cancer

DNA/RNA methylation plays an important role in lung cancer initiation and progression. Liquid biopsy makes use of cells, nucleotides and proteins released from tumor cells into body fluids to help with cancer diagnosis and prognosis. Methylation of circulating tumor DNA (ctDNA) has gained increasing attention as biomarkers for lung cancer. Here we briefly introduce the biological basis and detection method of ctDNA methylation, and review various applications of methylated DNA in body fluids in lung cancer screening, diagnosis, prognosis, monitoring and treatment prediction. We also discuss the emerging role of RNA methylation as biomarkers for cancer.     

An optimized rapid bisulfite conversion method with high recovery of cell-free DNA

Background

Methylation analysis of cell-free DNA is a encouraging tool for tumor diagnosis, monitoring and prognosis. Sensitivity of methylation analysis is a very important matter due to the tiny amounts of cell-free DNA available in plasma. Most current methods of DNA methylation analysis are based on the difference of bisulfite-mediated deamination of cytosine between cytosine and 5-methylcytosine. However, the recovery of bisulfite-converted DNA based on current methods is very poor for the methylation analysis of cell-free DNA.

Results

We optimized a rapid method for the crucial steps of bisulfite conversion with high recovery of cell-free DNA. A rapid deamination step and alkaline desulfonation was combined with the purification of DNA on a silica column. The conversion efficiency and recovery of bisulfite-treated DNA was investigated by the droplet digital PCR. The optimization of the reaction results in complete cytosine conversion in 30 min at 70 °C and about 65% of recovery of bisulfite-treated cell-free DNA, which is higher than current methods.

Conclusions

The method allows high recovery from low levels of bisulfite-treated cell-free DNA, enhancing the analysis sensitivity of methylation detection from cell-free DNA.