高通量测序技术在循环肿瘤DNA检测中的应用

目的 肿瘤是影响人类生命的重要威胁因素,肿瘤的早期诊断、预后及疗效评估有利于提高患者的生存率.目前临床上癌症的伴随诊断主要依靠肿瘤组织病理、内窥镜等检查,但都存在侵入性和异质性的问题.而在肿瘤细胞DNA中发生的分子变化如突变、易位和甲基化等,在循环肿瘤DNA(circulating tumor DNA,ctDNA)中也...     

循环肿瘤DNA检测及其临床应用研究进展

循环肿瘤DNA(ctDNA)主要来自于坏死、凋亡的肿瘤细胞,循环肿瘤细胞,肿瘤细胞分泌的外排体.ctDNA检测标本易获取、创伤小、方便动态监测,可反应肿瘤细胞基因改变的相关情况.ctDNA检测方法根据目标不同可分为两大类,一类是有针对性的检测特定的基因和突变,另一类是以非靶向方法检测所有的基因,通过与已知参考序列对照可...     

循环肿瘤DNA在胰腺癌诊治研究进展

胰腺癌是一种预后极差,严重威胁人类健康的恶性肿瘤.影像学、血清糖类抗原19?9(CA19?9)和组织活检等在胰腺癌的应用中都存在一定的局限性.循环肿瘤DNA(ctDNA)作为新兴的液体活检技术之一,因其标本采集的简便、非侵入性和实时反映肿瘤状态的特性,在胰腺癌的精准治疗中显示出巨大的应用前景.本文就ctDNA在胰腺癌的...     

循环肿瘤DNA的临床应用及展望

<正>癌症严重威胁着人类的生命健康。中国人口众多,医疗设备及医护人员配置暂时无法与发达国家相比,大部分癌症患者在确诊时病情已经进展到中晚期,错过了最佳的治疗时机。随着医学水平的发展和提高,人们已经意识到有同样病症的患者对于同样的治疗手段的获益各异。导致患者获益效果不同的原因有多种,解决问题的关键在于以更精细的手段对癌症进行分类,针对每个     

循环肿瘤RNA在肿瘤临床诊疗中的应用进展

液体活检是近年来研究的热点和难点,它能够非侵入性地反映体内肿瘤状态及预后情况。其中,新近兴起的一种标志物——循环肿瘤RNA（ctRNA）能够反映肿瘤来源的遗传信息,为肿瘤早期诊断、靶向药物使用以及预后分析提供有力依据。现阶段已有ctRNA检测试剂盒获准进入临床,例如微小RNA（miRNA）用于肝癌的辅助诊断等,越来越多...     

循环肿瘤DNA检测在非小细胞肺癌早期预警及监测管理中的研究进展

非小细胞肺癌(non-small cell lung cancer,NSCLC)约占肺癌的85%,发病率和病死率位居全球前列,由于其起病与进展的隐匿性,被发现时患者已丧失最佳的治疗时机。国内外课题组致力于NSCLC早期检测技术的研发及精准化治疗方法学的探寻,以期延长患者的无进展生存期和总存活时间。液体活检即通过血液、尿液或唾液等进行疾病特异性生物学标志物检测,具有微创性、高敏感性、可重复性等优势。该手段有助于癌症基因图谱的全面反映,极大克服了病灶组织学活检在时空上的异质性,对耐药靶点的发现、新药疗效预测及临床用药方案的调整意义深远。本文就液体活检中的环肿瘤DNA(circulating tumor DNA,ctDNA)检测在NSCLC早期诊断及监测管理的研究进展进行综述,以促进该技术临床应用价值的开拓。     

循环miRNA:潜在的肿瘤标志物

在最新一期的美国<国家科学院院刊>上,报道一则振奋人心的消息:验血可比CT扫描提前28个月发现肺癌.据称美国俄亥俄州大学综合癌症中心的研究人员通过分析肺癌患者血浆中miRNA表达谱,从而了解肿瘤患者疾病的状况及其侵袭性[1].全球每年约有700万人死于肿瘤,肿瘤的早期诊断和早期治疗对患者的预后生存率是极其重要的.临床上急切需要寻找灵敏度高和特异强的生物标志物,以便于对肿瘤进行早期诊断、鉴别分期、预后判断和疗效监测等.最近的研究发现,人类外周血中存在一些稳定性良好的循环miRNA,并且肿瘤患者血液中存在特异性的miRNA表达谱.因此,循环miRNA很可能成为肿瘤诊断的新型生物标志物.     

循环circRNA作为肺癌液体活检标志物的研究进展

肺癌是一种发病率和死亡率较高的恶性肿瘤,亟需新型有效的液体活检标志物以补充现有血液学诊疗指标的不足,改善肺癌患者的临床诊疗效果.循环circRNA是液体活检的一个重要组成部分,在肿瘤的形成和病情进展过程中发挥关键作用.研究表明,循环circRNA可作为肺癌诊断、疗效监测标志物及潜在的治疗靶点,是肿瘤液体活检领域的理想候...     

单细胞测序技术及其在肿瘤研究和临床诊断中的应用

肿瘤组织的异质性是肿瘤的一个重要特征.肿瘤细胞均一性的缺乏使得肿瘤靶向治疗的难度加大.因此,鉴别肿瘤细胞的异质性是一个重要工作.单细胞测序技术(single-cell sequencing,SCS)是通过下一代测序手段观察单个细胞的基因组序列信息,从而获得细胞间遗传物质和蛋白质信息的差异,因此能够更好地理解单个细胞在其微环境中的功能.通过对单个肿瘤细胞全基因组、转录组以及细胞表观遗传基因进行测序,瘤内异质性的复杂机制可被揭示,从而改善肿瘤诊断、转归预测和治疗效果的监测.本文对单细胞测序技术的发展、检测原理和检测流程、存在的问题及其在肿瘤研究中的应用以及该技术在临床上的潜在价值进行了概述.     

DNA甲基化与人类肿瘤

DNA甲基化不仅是最常见的真核DNA的修饰方式 ,而且正常的甲基化还维持着细胞和器官的正常功能。如果正常的甲基化模式遭到破坏 ,就会导致一些疾病的发生 ,如智力发育迟缓、免疫缺陷、散发性或遗传性肿瘤等。无论是生长调节基因的异常失活 ,还是整个染色体的去稳定 ,这些异常甲基化的无序状态都能促进肿瘤细胞的形成。一些基因的异常甲基化还与肿瘤化疗效果和患者的生存期直接相关 ,因此甲基化与人类肿瘤的关系越来越受到人们的重视。一、DNA甲基化在哺乳类动物中 ,胞嘧啶的甲基化是惟一已知的DNA内源性修饰。通过酶催化作用 ,甲基化基…     

Relative exchangeable copper: a new highly sensitive and highly specific biomarker for Wilson's disease diagnosis

Wilson disease (WD) is an autosomal recessive inherited disorder of copper metabolism. Failure to diagnose WD can be dramatic leading to irreversible damages. The molecular genetic analysis of ATP7B gene is the reference test for diagnosis but the number of reported mutations of the ATP7B gene is on the rise. The analysis is cumbersome and requires tedious work. Other clinical and biological tests are proposed but it is often difficult to interpret some patients' results. A rapid and reliable biological test for WD diagnosis is still needed. Analytical reliability of Exchangeable copper (CuEXC) determination procedure is examined by studying the repeatability, the short term stability and stability in frozen serum. Relative exchangeable copper (REC=CuEXC/total copper%) is proposed and evaluated as a new diagnostic test and compared to classic tests used for WD diagnosis. Sixteen new Wilson disease patients were diagnosed in our institution between January 2009 and May 2011. The different biological tests used for WD diagnosis yielded lower sensitivity and specificity compared to our new biomarker, the REC. We show that REC is an excellent discriminatory tool for the diagnosis of WD offering 100% sensitivity and 100% specificity.     

Development of a sensitive and specific enzyme-linked immunosorbent assay for thymosin beta15, a urinary biomarker of human prostate cancer

OBJECTIVES: In tissue-based assays, thymosin beta15 (Tbeta15) has been shown to correlate with prostate cancer (CaP) malignancy and with future recurrence. To be clinically effective, it must be shown that Tbeta15 is released by the tumor into body fluids in detectable concentrations. Toward this end, we have worked to develop a quantitative high-throughput assay that can accurately measure clinically relevant concentrations of Tbeta15 in human urine. DESIGN AND METHODS: Sixteen antibodies were raised against recombinant Tbeta15 and/or peptide conjugates. One antibody, having stable characteristics over the wide range of pH and salt concentrations found in urine and minimal cross-reactivity with other beta thymosins, was used to develop a competitive enzyme-linked immunosorbent assay (ELISA). Urinary Tbeta15 concentration was determined for control groups; normal (N = 52), prostate intraepithelial neoplasia (PIN, N = 36), and CaP patients; untreated (N = 7) with subsequent biochemical failure, radiation therapy (N = 17) at risk of biochemical recurrence. RESULTS: The operating range of the competition ELISA fell between 2.5 and 625 ng/mL. Recoveries exceeded 75%, and the intra- and inter-assay coefficients of variability were 3.3% and 12.9%, respectively. No cross-reactivity with other urine proteins was observed. A stable Tbeta15 signal was recovered from urine specimens stored at -20 degrees C for up to 1 year. At a threshold of 40 (ng/dL)/mug protein/mg creatinine), the assay had a sensitivity of 58% and a specificity of 94%. Relative to the control groups, Tbeta15 levels were greater than this threshold in a significant fraction of the CaP patients (P < 0.001), including 5 of the 7 patients who later experienced PSA recurrence. CONCLUSIONS: We have established an ELISA that is able to detect Tbeta15 at clinically relevant concentrations in urine from patients with CaP. The assay will provide a tool for future clinical trials to validate urinary Tbeta15 as a predictive marker for recurrent CaP.     

A sensitive enzyme immunoassay specific for human chorionic gonadotrophin

A sensitive enzyme immunoassay procedure for the β subunit of HCG is described. The procedure should be suitable for the early diagnosis of pregnancy, the management of threatened abortion, and as a tumour marker in patients with trophoblastic neoplasms.     

A sensitive and specific sandwich enzyme immunoassay for human thyroid-stimulating hormone

A sensitive and specific sandwich enzyme immunoassay (EIA) for human thyroid-stimulating hormone (hTSH) has been developed. hTSH is incubated with anti-hTSH IgG-coated polystyrene balls, and after washing they are further incubated with anti-hTSH Fab'-β-d-galactosidase conjugate. The β-d-galactosidase activity bound to the polystyrene balls is proportional to the amount of hTSH to be assayed. Polystyrene balls are coated with rabbit anti-hTSH IgG which had been affinity-purified and treated with human chorionic gonadotropin-Sepharose 4B to remove antibodies cross-reacting with structurally related hormones. Rabbit anti-hTSH Fab', which had been affinity-purified was conjugated with β-d-galactosidase from Escherichia coli using N,N′-o-phenylenedimaleimide.In the specific sandwich enzyme immunoassay developed, 1 nU (1 × 10^?9 U) of hTSH per tube can be measured and the sensitivity for serum hTSH level is 0.1 μU/ml when 10 μl of serum is used. No significant interference was observed in the presence of 1.3 mU hLH/tube, 0.5 mU hFSH/tube or 0.5 U hCG/tube. Recoveries of hTSH added to human sera were 95.3–104% with a standard deviation of 12.0–14.9%. The coefficients of within-assay and between-assay variations were 6.0–7.5% and 4.9–8.7%, respectively. The regression equation and coefficient for correlation to radioimmunoassay (RIA) were y (RIA) = 0.95x (EIA) + 3.2 and 0.97, respectively.Serum levels of hTSH in normal male and female adults were 2.4 ± 1.0 (SD) (n = 41) and 2.9 ± 1.3 (n = 46) μU/ml, respectively; those in hyperthyroidism and hypothyroidism were 0.28 ± 0.06 (n = 20) and 49.6 ± 24.7 (n = 22) μU/ml, respectively; and those in pregnant and postmenopausal women were 2.5 ± 1.2 (n = 7) and 2.7 ± 1.0 (n = 35) μU/ml, respectively, indicating that high serum levels of hCG or hLH and hFSH under these conditions did not significantly interfere with the present assay of hTSH at normal levels.     

Current concepts in biomarker technology for bladder cancers

BACKGROUND: Transitional cell carcinoma of the bladder (TCC) is the second most common malignancy of the urinary tract. More than 70% of treated tumors recur, and 30% of recurrent tumors progress. Currently, pathologic staging and grading are valuable prognostic factors for detecting and monitoring TCC. Urinalysis, cystoscopy, and cytology are either invasive or lack sensitivity and specificity. The availability of a noninvasive, reliable, and simple test would greatly improve the detection and monitoring of patients with TCC. Several biomarkers for bladder cancer have been proposed, but no single marker has emerged as the test of choice. APPROACH: We undertook a comprehensive literature search using Medline to identify all publications from 1980 to 1999. Articles that discussed potential biomarkers for TCC were screened. Only compounds that demonstrated high sensitivity or specificity, significant correlation with TCC diagnosis and staging, and extensive investigation were included in this review. CONTENT: Potential biomarkers of disease progression and prognosis include nuclear matrix protein, fibrin/fibrinogen product, bladder tumor antigen, blood group-related antigens, tumor-associated antigens, proliferating antigens, oncogenes, growth factors, cell adhesion molecules, and cell cycle regulatory proteins. The properties of the biomarkers and the methods for detecting or quantifying them are presented. Their sensitivities and specificities for detecting and monitoring disease were 54-100% and 61-97%, respectively, compared with 20-40% and 90% for urinalysis and cytology. SUMMARY: Although urine cytology and cystoscopy are still the standard of practice, many candidate biomarkers for TCC are emerging and being adopted into clinical practice. Further research and better understanding of the biology of bladder cancer, improved diagnostic techniques, and standardized interpretation are essential steps to develop reliable biomarkers. It is possible that using the current biomarkers as an adjuvant modality will improve our ability to diagnose and monitor bladder cancer.     

Establishing a sensitive and specific assay for determination of glucocorticoid bioactivity

Glucocorticoids are hormones that play a major role in energy homeostasis and stress response of the body. As drugs they are most frequently used for immunosuppressive and anti-inflammatory purposes. Glucocorticoids are exploited successfully in the treatment of a wide variety of diseases; however, some patients develop side-effects, while others fail to respond to this form of therapy. Alterations in pharmacodynamic and pharmacokinetic actions might contribute to individual differences in glucocorticoid sensitivity. Antibody-based methods such as RIA (Radioimmunoassay) and ELISA (Enzyme-linked immunosorbent assay) are routinely used to determine glucocorticoid serum levels. However, as these techniques measure the total amount of a specific glucocorticoid and do not discriminate between protein-bound and freely available (i.e. biologically active) glucocorticoids, the results do not necessarily reflect the active levels of glucocorticoid, i.e. the "glucocorticoid milieu" in a patient. Being able to determine glucocorticoid bioactivity in serum or other body fluids could help identifying glucocorticoid-sensitive or -resistant patients and help finding explanations for different responses in individual patients. For this reason, we established a glucocorticoid bioactivity assay that is based on the measurement of glucocorticoid-dependent reporter gene activity. Making use of a human T-cell leukemia line, equipped with the glucocorticoid receptor and the fluorescence protein Venus as the assay's reporter (Jurkat(GR)-MMTV-VNP), glucocorticoid bioactivity can be determined from small amounts of serum or other biologic fluids. The developed glucocorticoid bioassay is both sensitive and reproducible, without any relevant cross-reactivity with steroid hormones other than glucocorticoids and can be practically applied in daily laboratory routine.     

An N-terminal truncated carboxypeptidase E splice isoform induces tumor growth and is a biomarker for predicting future metastasis in human cancers

Metastasis is a major cause of mortality in cancer patients. However, the mechanisms governing the metastatic process remain elusive, and few accurate biomarkers exist for predicting whether metastasis will occur, something that would be invaluable for guiding therapy. We report here that the carboxypeptidase E gene (CPE) is alternatively spliced in human tumors to yield an N-terminal truncated protein (CPE-ΔN) that drives metastasis. mRNA encoding CPE-ΔN was found to be elevated in human metastatic colon, breast, and hepatocellular carcinoma (HCC) cell lines. In HCC cells, cytosolic CPE-ΔN was translocated to the nucleus and interacted with histone deacetylase 1/2 to upregulate expression of the gene encoding neural precursor cell expressed, developmentally downregulated gene 9 (Nedd9)--which has been shown to promote melanoma metastasis. Nedd9 upregulation resulted in enhanced in vitro proliferation and invasion. Quantification of mRNA encoding CPE-ΔN in HCC patient samples predicted intrahepatic metastasis with high sensitivity and specificity, independent of cancer stage. Similarly, high CPE-ΔN mRNA copy numbers in resected pheochromocytomas/paragangliomas (PHEOs/PGLs), rare neuroendocrine tumors, accurately predicted future metastasis or recurrence. Thus, CPE-ΔN induces tumor metastasis and should be investigated as a potentially powerful biomarker for predicting future metastasis and recurrence in HCC and PHEO/PGL patients.     

Forkhead box F2 as a novel prognostic biomarker and potential therapeutic target in human cancers prone to bone metastasis: a meta-analysis

ObjectiveTo undertake a systematic review and meta-analysis to evaluate the prognostic value of Forkhead box F2 (FOXF2) levels in different types of cancers prone to bone metastasis.MethodsA systematic search of publications listed in electronic databases (The Web of Science, EMBASE®, PubMed®, PMC, Science Direct and CNKI) from inception to 5 November 2020 was conducted. The hazard ratios (HRs) and 95% confidence intervals (95% CIs) were used to assess the relationship between FOXF2 levels and patient prognosis including overall survival (OS) and disease-free survival (DFS).ResultsSixteen studies enrolling 8461 participants were included in the meta-analysis. High levels of FOXF2 were a predictor of OS (HR: 0.66; 95% CI 0.51, 0.86) and DFS (HR: 0.60; 95% CI 0.48, 0.76). The trim-and-fill analysis, sensitivity analysis and subgroup analyses stratified by the study characteristics confirmed the robustness of the results.ConclusionThese current findings indicate that high FOXF2 levels could be an indicator of a good prognosis in cancer patients with tumours that are prone to bone metastasis. FOXF2 levels might be a clinically important prognostic biomarker.     

A sensitive and specific fluorimetric method for the determination of noradrenalin and adrenalin in human plasma

A sensitive and specific fluorimetric method which allows measurement of plasma noradrenalin and adrenalin concentrations even at physiological levels is described. A two-step Chromatographie separation on alumina and Amberlite CG-50 (Na⁺ form) is employed, perchloric and boric acids being used for elution. Fluorophores of increased fluorescence intensity and stability are obtained by using a dimercaptopropanol-formaldehyde solution as antioxidant.

The smallest amount of noradrenalin or adrenalin which would double blank values is about 50 picograms. When this procedure is applied to 10 ml venous plasma of normal subjects at rest, fluorimetric readings of the samples, 4–10 times blank values are obtained and excitation spectra show the typical morphology of noradrenalin and adrenalin fluorophores. Fluorimetric discrimination allows sufficiently accurate differential estimation of mixtures of the two amines in 1:25 proportion. Normal values in peripheral venous plasma for noradrenalin were 174 ± 24 ng/l and for adrenalin 66 ± 13 ng/l (mean ± S.D.).