外周血液中循环肿瘤细胞和循环游离DNA检测在乳腺癌患者中的应用

目的 探究外周血液中循环肿瘤细胞(CTCs)和循环游离DNA(cfDNA)检测在乳腺癌患者中的应用效果.方法 收集本院2018-02-2019-02收治的94例乳腺癌患者、48例乳腺良性疾病患者及56例健康体检者外周血液标本,采用基于尺寸的高通量微流控芯片捕获CTCs、基于Alu序列的实时荧光定量PCR检测游离DNA长...     

前列腺癌患者外周血PSA mRNA的意义

目的 探讨前列腺特异抗原（PSA） mRNA在前列腺癌（PCa)患者外周血表达的意义。方法 运用巢式RT－PCR检测36例PCa患者,28例良性前列腺组织增生（BPH）患者和15例健康志愿者外周血。结果 BPH患者和志愿者外周血PSA mRNA均阴性,36例PCa外周血PSA mRNA阳性率38．9％（14／36）。PSA mRNA表达阳性率随肿瘤分期进展而增高,各期阳性率有显著差异（P＜0．05）。结论 PSA mRNA特异表达于PCa外周血,可用作PCa微转移的指标。     

大肠癌外周血循环肿瘤细胞数的检测及临床意义

目的 检测大肠癌（CRC）患者外周血循环肿瘤细胞（CTCs）数,探讨其临床意义.方法 58例CRC患者术前静脉采血,使用多重聚合酶链式反应（PCR）结合荧光定量PCR（RTFQ PCR）方法检测外周血细胞角蛋白20（CK20）及癌胚抗原（CEA） mRNA表达,比较CTCs细胞阳性率与临床病理参数的关系.结果 58例CRC患者外周血中CK20及CEAmRNA表达的阳性率为93.1％.CTCs的阳性检出率与Ducke＇s分期及淋巴结转移相关（P＜0.05）.结论 多重RTFQ PCR方法可应用于CRC患者外周血中CTCs检测,对预后判断有着重要的临床意义.     

外周血循环肿瘤DNA检测方法及应用

肿瘤是一种威胁人类健康的重大疾病.研究发现,肿瘤的发生和发展是基于分子水平上癌基因(oncogenes)的激活和抑癌基因(anti-oncogene)的失活所致.基因突变分析对于肿瘤诊断和预后至关重要.早期诊断被认为是提高癌症病人治愈率,降低其死亡率最有效的途径.外周血循环肿瘤核酸中可检测到与肿瘤细胞一致的基因突变.这些基因突变的检测可以为肿瘤的分期及药物的治疗疗效和预后提供丰富的信息.因此,外周血循环肿瘤DNA突变检测为肿瘤的早期诊断和治疗提供了新的研究方向和领域.     

食管癌患者循环DNA的定量分析及其临床应用价值

目的探讨食管癌患者循环DNA的来源、临床应用价值及可能的影响因素。方法收集53例食管癌患者术前和术后2h的静脉血,用SYBRgreenⅠ荧光染色法和检测CEA-mRNA表达的实时荧光定量RT-PCR法,分别检测血浆中的DNA水平和循环中的肿瘤细胞数。结果食管癌组术前循环DNA水平为(67.46±59.78)μg/L,显著高于良性肿瘤组(23.52±22.94)μg/L和健康组(11.97±12.35)μg/L,均P<0.001,ROC曲线下的面积为0.798。术后DNA水平迅速上升,达(122.36±71.41)μg/L,显著高于手术前(P<0.001)。CEAmRNA 循环肿瘤细胞数在术后也显著高于手术前,其中位数水平分别为1513(95%CI,660~7974)/ml全血和188(95%CI,155~498)/ml全血,P<0.01。血浆DNA水平与循环肿瘤细胞数之间,无论在术前还是术后均无显著性关联(r=0.200,P=0.156和r=0.206,P=0.138),与临床病理分期也无相关性(r<0.030,P>0.05)。结论食管癌患者循环DNA水平增高,可为疾病诊断提供一定的辅助信息,但没有足够的敏感性和特异性成为诊断或监测癌症的独立标志物。     

The potential of circulating cell free RNA as a biomarker in cancer

Introduction: It is now clear that circulating cell-free ribonucleic acids (ccfRNAs), including messenger RNA (mRNA) and miRNA, are potential cancer biomarkers. As ccfmiRNA is relatively more stable than ccfmRNA, research should concentrate on developing novel methods to preserve the stability of ccfmRNA and standardization of the protocol which includes extraction, detection, and multicenter validation.

Areas covered: This literature review concentrates on the potential of ccfRNA being used as a biomarker in cancer, with special focus on mRNAs and microRNAs (miRNAs).

Expert opinion: With the advancement of high-throughput technologies such as RNA sequencing, a panel of biomarkers will be used for the diagnosis, prognosis and therapeutic monitoring of cancer patients. In order to achieve this important target, bioinformatics education to pathologists, scientists, and technologists in molecular diagnostic laboratories is essential. Moreover, the panel of these new ccfRNAs biomarkers has to obtain approval or clearance from an authority such as the US Food and Drug Administration (FDA), and the standard of utilizing these new protocols has to be recognized via accreditation exercise. Therefore, there is still a long way to go before an extensively use of ccfRNA biomarkers in cancer patients can be realized.     

Isolation of rare circulating tumor cells in cancer patients: technical aspects and clinical implications

Circulating tumor cells (CTCs) may be detected in the blood of patients with epithelial tumors using different analytical approaches. The relative number of CTCs is low and they include a heterogeneous population of cells with diverse biological and molecular characteristics, often different from those of the respective primary tumor. Until recently, they have been difficult to detect and, even though discordant results have been reported when different methods of detection were used, they may provide prognostic and predictive information. Several antibody- or molecular-based CTC detection methods have been developed, offering hope for individualized risk assessment by utilizing CTCs as biomarkers of disease progression and drug response. Pilot studies have also shown that by utilizing methods that permit, besides enumeration, a molecular characterization of CTCs, one could better identify high-risk patients, predict response to targeted therapies, analyze gene expression profiles (in order to identify new potential drug targets) and increase our knowledge of the metastatic process. In this article we review the techniques currently utilized for isolation and characterization of CTCs and we discuss their potential utility in clinical oncology focusing on the future perspectives in this field.     

专用采血管对血浆中循环游离DNA保存效果的影响

目的 验证专用采血管对血浆中循环游离DNA(cfDNA)保存效果的影响.方法 分别用EDTA-K2抗凝管和两种cfDNA保存专用采血管采集健康成人外周血,待血样室温保存到固定的时间点时,分离提取血浆中的cfDNA,定量检测不同时间点血浆中cfDNA水平的变化情况.结果 采集在EDTA-K2抗凝管中的血浆cfDNA水平随...     

前列腺癌患者外周血DD3 mRNA的检测及临床意义

目的　探讨检测前列腺癌(PC)患者外周血DD3mRNA在诊断和治疗监测中的意义。方法　用巢式逆转录聚合酶链反应(nRT PCR)检测44例不同分期PC患者和30例良性前列腺增生(BPH)患者及18名健康男性志愿者外周血中DD3mRNA,并进行对比分析。结果　BPH患者和健康男性志愿者外周血中DD3mRNA均阴性;未治疗PC组外周血DD3mRNA100%阳性(11 /11),而经内分泌治疗PC组阳性率30. 3% ( 10 /33 ),两组差异有统计学意义(P<0. 01 )。结论　外周血DD3mRNA是PC诊断和内分泌治疗监测的良好指标,有望成为PC特异性肿瘤标志物。     

Diagnostic potential of circulating cell-free DNA in patients needing mechanical ventilation: promises and challenges

Circulating cell-free DNA (cf-DNA) mainly comes from apoptotic cells and can reflect the extent of cellular damage. Increased plasma levels of cf-DNA have been found in many acute disorders, including septic and clinically ill patients, and usually correlate well with clinical outcome. Acute respiratory failure, the most frequent organ failure in ICU patients, can be related to various acute diseases that may cause cell death and release of DNA into the bloodstream. In a recent issue of Critical Care, Okkonen and colleagues evaluate levels of cf-DNA in plasma as a prognostic marker in patients needing mechanical ventilation. They report that plasma cf-DNA was higher than normal in patients with mechanical ventilation, and even higher in patients who eventually died compared to survivors. However, its usefulness as a death predictor may be limited in the heterogeneous group of mechanically ventilated patients, probably due to confounding effects of co-morbidities, among other factors.     

前列腺癌影像诊断现状及新进展

随着前列腺癌发病率的上升,早期诊断及分期从而指导临床治疗一直是影像学研究的重点。目前影像学技术发展迅猛,前列腺癌影像诊断新技术和新方法不断出现。现将前列腺癌的各种影像学检查技术及其新进展进行综述。     

The implications of improvements in screening for prostate cancer

With Men's Health Week running from 10 to 16 June this year, there is an increasing focus on men's health. Prostate cancer may be traditionally associated with elderly men, but with improvements in screening techniques increasing numbers of patients are being diagnosed at a younger age, perhaps changing the view of this disease.     

Detection of Mammaglobin A-mRNA-positive circulating tumor cells in peripheral blood of patients with operable breast cancer with nested RT-PCR

OBJECTIVES: The development and validation of a nested RT-PCR methodology for the detection of Mammaglobin A-mRNA-positive circulating tumor cells in peripheral blood of patients with operable breast cancer and evaluation of its prognostic significance. DESIGN AND METHODS: Different combinations of specific primers were in silico designed and selected, so that false positive results due to genomic DNA contamination were avoided. The specificity of the primers used was evaluated in 30 healthy individuals, 20 patients with colorectal cancer and 20 patients with non-small cell lung cancer. The method was applied in 101 patients with operable breast cancer before the administration of adjuvant chemotherapy and 39 patients with metastatic breast cancer. RESULTS: Mammaglobin A-mRNA-positive cells were detected in 14/101 (13.9%) of early breast cancer patients but not in the control population studied (0%); 9 of them (64.3%) relapsed during the follow-up period. Mammaglobin A was detected in 7/39 (17.9%) of patients with verified metastasis. Multivariate analysis revealed the detection of Mammaglobin A-mRNA-positive cells, as an independent risk factor for reduced DFI. CONCLUSIONS: Mammaglobin A is a highly specific molecular marker for the detection of circulating tumor cells in operable breast cancer, with important prognostic applications.     

Increased integrity of free circulating DNA in sera of patients with colorectal or periampullary cancer: direct quantitative PCR for ALU repeats

BACKGROUND: Cell-free DNA circulating in blood is a candidate biomarker for malignant tumors. Unlike uniformly truncated DNA released from apoptotic nondiseased cells, DNA released from dead cancer cells varies in size. We developed a novel method to measure the ratio of longer to shorter DNA fragments (DNA integrity) in serum as a potential biomarker for patients with colorectal cancer (CRC) or periampullary cancers (PACs). METHODS: Sera from 32 patients with CRC (3 stage I, 14 stage II, 6 stage III, and 9 stage IV patients), 19 patients with PACs (2 stage I, 9 stage II, 1 stage III, and 7 stage IV patients), and 51 healthy volunteers were assessed by quantitative real-time PCR of ALU repeats (ALU-qPCR) with 2 sets of primers (115 and 247 bp) amplifying different lengths of DNA. We used serum directly as a template for ALU-qPCR without DNA purification. DNA integrity was determined as ratio of qPCR results of 247-bp ALU over 115-bp ALU. RESULTS: ALU-qPCR had a detection limit of 0.01 pg of DNA. Eliminating DNA purification reduced technical artifacts and reagent/labor costs. Serum DNA integrity was significantly increased for stage I/II and III/IV CRC and stage I/II and III/IV PACs (P = 0.002, P = 0.006, P = 0.022, and P <0.0001, respectively). ROC curves for detecting CRC and PACs had areas under the curves of 0.78 and 0.80, respectively. CONCLUSIONS: Direct ALU-qPCR is a robust, highly sensitive, and high-throughput method to measure serum DNA integrity. DNA integrity is a potential serum biomarker for detection and evaluation of CRC and PACs.     

非小细胞肺癌患者外周血循环内皮干细胞的检测及其临床意义

目的探讨外周血循环内皮祖细胞（endothelial progenitors cells,EPCs）与非小细胞肺癌患者的关系及临床意义。方法选择非小细胞肺癌患者45例和对照组15例,用抗CD133和KDR的单克隆抗体标记外周血细胞,应用流式细胞术检测其外周血中循环内皮祖细胞。应用Real-time RT-PCR方法检测患者外周血中CD133和KDR的mRNA表达水平。密度梯度离心法从人外周血分离出单个核细胞,将其接种在人纤维粘连蛋白包被培养板,在加有EGM-2-MV-SingleQuots的培养液中培养,在荧光显微镜下进行鉴定。其中U EA-1和D iI-A c-LDL染色双阳性细胞为正在分化的内皮祖细胞。结果肿瘤患者外周血中EPCs含量高于正常对照组（P〈0.01）,外周血中CD34和KDR的表达水平明显高于正常对照组。结论非小细胞肺癌患者外周血EPCs明显增高,为今后将EPCs作为非小细胞肺癌患者辅助诊断和治疗效果检测的标志物奠定基础。     

前列腺癌患者外周血中前列腺癌抗原3基因和前列腺特异性抗原基因联合检测的意义

目的 评价外周血前列腺痛抗原3基因(prostate cancer antigen 3,PCA3 mRNA)和前列腺特异性抗原基因(prostate specific antigen,PSA mRNA)联合检测对前列腺癌(PCa)及对其微转移诊断的价值.方法 用双重荧光实时定量逆转录(dFQ-RT)-PCR对49例PCa和71例前列腺增生(BPH)患者外周血PCA3 mRNA和PSA mRNA进行定量检测,通过受试者工作特征(ROC)曲线评价其在PCa诊断和微转移监测中的价值.结果 PCa组外周血PCA3 mRNA含量明显高于BPH组[2 362(<3-7 421)拷贝/ml比<30拷贝/ml,Z=-6.66,P<0.01],而PSA mRNA含量也明显高于BPH组[3 425(908～36 639)拷贝/ml比<200拷贝/ml,Z=-6.40,P<0.01];PCa组外周血PCA3mRNA和PSA mRNA的阳性率随临床分期增高而增加[B期:均为30.0%(3/10),C期:60.0%(9/15)和86.7%(13/15),D期:91.7%(22/24)和91.7%(22/24);χ2分别为13.534和16.451,P均<0.01],同时也随Gleason评分增高而增加[2～4分:20.0%(1/5)和40.0%(2/5);5～7分:66.7%(12/18)和72.2%(13/18);8～10分:84.6%(22/26)和92.3%(24/26);χ2分别为8.895和8.015,P均<0.05];ROC曲线显示,当PCA3 mRNA和PSA mRNA临界值分别为846拷贝/ml和280拷贝/ml时,诊断PCa敏感度分别为69.4%(34/49)和81.7%(40/49),特异度分别为90.1%(64/71)和77.5%(55/71);而联合检测时其敏感度可增至85.7%(42/49),但特异度下降为76.1%(54/71).PCA3 mRNA诊断PCa微转移的敏感度和特异度分别为90.9%(20/22)和84.7%(11/13).结论 外周血PCA3 mRNA和PSA mRNA检测是PCa诊断的良好指标,而联合检测可弥补PCA3 mRNA敏感度低和PSA mRNA特异度低的不足,而更有利于PCa诊断;PCA3 mRNA可能为PCa微转移诊断的良好指标.