前列腺癌组织中GSTP1和DAPK基因异常甲基化检测及临床意义

目的:检测前列腺癌组织中GSTP1和DAPK基因异常甲基化,探讨其甲基化与前列腺癌临床病理特征间的关系.方法:采用巢式甲基化特异性PCR(nested methylation specific polymerase chain reaction,NMSP)法对57例前列腺癌组织和35例良性前列腺增生组织进行甲基化检测,分析其甲基化的发生与前列腺癌临床病理特征间的关系.结果:前列腺癌组织中GSTP1和DAPK基因甲基化检出率显著高于良性前列腺增生组织(GSTP1:61.4%vs 2.9%;DAPK:43.9%vs 8.6%;均P＜0.01).GSTP1基因甲基化率在不同Gleason评分间具有明显差异(x2=11.53,P=0.001),而在不同年龄、前列腺特异性抗原(PSA)和临床分期之间差异却无显著性(x2=0.111,1.662和1.975,均P＞0.05);DAPK基因甲基化率在不同年龄、PSA、Gleason评分和不同临床分期之间均无明显差异(x2=0.071、0.002、1.290和2.309,P均＞0.05).结论:GSTP1和DAPK基因异常甲基化与前列腺癌的发生与发展相关,可有助于前列腺癌的诊断.     

前列腺癌组织中DLC-1启动子甲基化定量检测的HRM方法建立及其应用意义

目的 建立HRM技术定量检测DLC-1启动子甲基化的方法 ,并分析探讨DLC-1甲基化程度与前列腺癌病理参数的相关性.方法 选取89份前列腺癌组织以及10份匹配癌旁组织标本,采用LCM收集肿瘤细胞群,抽提DNA后进行甲基化修饰.以CpGenome Universal Methylated DNA作为100%甲基化样本,以100%非甲基化的健康人外周血DNA作为稀释剂,分别制成100%、80%、50%、30%、10%、0%系列浓度的标准曲线,并进行重复性和灵敏性评价.同时用HRM技术定量检测前列腺癌细胞中DLC-1甲基化水平,探讨DLC-1甲基化程度与前列腺癌患者年龄、PSA水平与前列腺癌TNM的关系.结果 100%、80%、50%、30%、10%、0%甲基化标准品的HRM熔解曲线从右往左依次排列,10份癌旁组织标本、35份前列腺癌组织标本重叠在0%标准曲线上;5份前列腺癌组织标本位于0%～30%区域内,29份位于31%～80%区域内,20份位于81%～100%区域内.HRM技术最低检测限可达10%的甲基化水平,优于MSP检测(30%甲基化水平).DLC-1甲基化水平与前列腺癌患者年龄无明显相关(X~2=3.29,P=0.19),与PSA水平也无相关性(X~2=2.04,P=0.36),但DLC-1甲基化水平与TNM分期显著相关(X~2=9.04,P=0.01),且随TNM分期增高而增高.结论 成功建立的HRM技术定量检测DLC-1甲基化水平的方法 稳定、灵敏、操作简单,DLC-1启动子甲基化有望成为评估前列腺癌TNM的分子指标.     

Detection of aberrant promoter methylation of GSTP1 in the tumor and serum of Chinese human primary hepatocellular carcinoma patients

PURPOSE AND EXPERIMENTAL DESIGN: Glutathione S-transferases, enzymes that prevent cells from damage mediated by oxidant and electrophilic carcinogens, may be early critical determinants of carcinogenesis. To explore the aberrant promoter CpG island methylation of the GSTP1 gene as a biomarker for screening hepatocellular carcinoma (HCC) high risk individuals and for the early detection of HCC, we analyzed its methylation in the tumor and non-tumor tissues and serum samples from 26 patients with HCC, as well as serum from 8 liver cirrhosis patients by methylation-specific PCR (MSP). RESULTS: Twenty-three of 26 (88.5%) tumor tissues and 18 of 26 (69%) corresponding non-tumor tissues displayed GSTP1 promoter CpG island hypermethylation. Similarly, GSTP1 promoter hypermethylation was detected for the first time in 16 of 32 (50%) of circulating tumor DNA in the peripheral serum from HCC patients and 4 of 8 (50%) cirrhosis tissues and 3 of 8 (37.5%) corresponding serum DNA from cirrhosis patients. The aberrant methylation of the GSTP1 gene in the serum of patients is in agreement with tumor methylation status (P = 0.004). None of the 12 normal PBMC samples were methylation positive. CONCLUSIONS: These data indicate that the epigenetic aberrance of promoter CpG island hypermethylation of the GSTP1 gene may contribute to the hepatopathogenesis of HCC and is a potential valuable biomarker for noninvasive disease monitoring and HCC early diagnosis.     

前列腺癌干细胞分离方法的对比及筛选

背景：前列腺癌干细胞是前列腺癌复发侵袭的重要原因,目前研究难点在于前列腺癌干细胞分离技术效率较低。目的：探索高效地从人前列腺癌PC-3及LNCap细胞株分离前列腺癌干细胞的方法。方法：采用含血清贴壁培养法及无血清悬浮培养法培养PC-3及LNCap细胞株,然后利用流式细胞表面标记CD133及CD44检测两种细胞在不同培养条件下可获取前列腺癌干细胞的比例,同时采用诱导分化实验初步鉴定前列腺癌干细胞特性。结果与结论：PC-3及LNCap细胞能在添加生长因子的无血清培养基中形成悬浮细胞球,接种在含血清培养基后可以诱导分化为贴壁细胞;无血清培养组的CD44^＋/CD133^＋细胞比例：PC-3为0.59%,LNCap为1.71%,含血清培养组中的CD44^＋/CD133^＋细胞比例：PC-3为0.32%,LNCap为0.73%,其中LNCap细胞采用两种方法所获的CD44^＋/CD133^＋细胞均高于PC-3所获的的细胞（P〈0.05）,在两种细胞中无血清悬浮培养和含血清贴壁培养差异无显著性意义（P〉0.05）,但无血清悬浮培养周期长,获得细胞数相对较少,直接影响分选后肿瘤干细胞功能测定。因此可以证实含血清贴壁培养LNCap细胞较无血清悬浮培养法更能高效快捷的获取前列腺癌干细胞。     

Lectin and serum-PSA interaction as a screening test for prostate cancer

OBJECTIVES: The present investigation was designed to distinguish prostate cancer and benign prostate hyperplasia by lectin-prostate specific antigen (PSA) binding. DESIGN AND METHODS: The quantitative precipitin method of concanavalin A (Con A)-carbohydrate interaction was explored with the serum PSA of patients suffering from prostatic complications. RESULTS: The carbohydrate content in the precipitate after binding of Con A with serum PSA of prostate cancer was significantly lower than that of benign prostate hyperplasia. This may be due to altered sugar chain structure or less glycosylation of PSA in prostate cancer. CONCLUSIONS: We conclude that a serum value <3.0 microg/ml of the carbohydrate content of Con A-PSA precipitate indicates strong suspicion for prostate cancer and this cut off level is effective in reducing the rate of unnecessary biopsies in men with total PSA value between 4.0 to 10.0 ng/ml.     

Prostate-specific antigen and screening for prostate cancer

This article discusses prostate-specific antigen (PSA) and screening for prostate cancer. Topics explored include the history of PSA testing, the biology of PSA, clinical uses of PSA testing, improving the accuracy of PSA testing, and controversies in prostate cancer screening.     

Prognostic significance of RASSF1A promoter methylation in operable breast cancer

ObjectivesThe aim of our study was to evaluate the prognostic significance of RASSF1A promoter methylation status in operable breast cancer.Design and methodsBy using Methylation Specific PCR, we evaluated the specificity of RASSF1A promoter methylation in 10 breast tumors and matching normal tissues, 10 breast fibroadenomas and 11 normal breast tissues. The prognostic significance of RASSF1A methylation was validated in 93 formalin fixed paraffin-embedded (FFPE) tissues obtained from patients with operable breast cancer.ResultsMethylation of RASSF1A promoter was observed in 1/31 (3.2%) non-cancerous breast tissues and 53/93 (57.0%) early stage breast tumors. The only positive sample in the non-cancerous breast tissues group was found in a histological normal tissue surrounding the tumor. During the follow-up period, 24/93 (25.8%) patients relapsed and 19/93 (20.4%) died. Disease-Free-Interval (DFI) was significantly associated with RASSF1A methylation (p = 0.028).ConclusionsRASSF1A promoter methylation provides important prognostic information in early stage breast cancer patients.     

人胃癌细胞系端粒酶逆转录酶基因启动子区域甲基化的检测

目的研究人胃癌细胞系端粒酶逆转录酶基因（hTERT）启动子区域是否存在甲基化,甲基化程度是否与肿瘤的恶性程度相关。方法运用甲基化特异性PCR（MSP）方法,以人乳腺癌细胞、人胚肺成纤维细胞作为阳性及阴性对照,检测中、低分化的胃腺癌细胞系hTERT基因启动子区域甲基化情况。结果端粒酶阳性的胃癌细胞、人乳腺癌细胞均存在甲基化,而端粒酶阴性的人胚肺成纤维细胞不存在甲基化,不同分化程度胃癌细胞的hTERT启动子区CpG位点甲基化情况不同。结论hTERT基因启动子区域的甲基化,与细胞的分化程度相关,可能参与了胃癌细胞的发生与发展。     

Real-time telomerase activity measurements for detection of cancer

Since the hallmark report of the PCR-based telomeric repeat amplification protocol (TRAP) in 1994, there has been a flurry of investigations of telomerase activity on normal, benign, premalignant and cancerous samples representative of the various stages of tumorigenesis. Basic research and technological advances in human genetics, biochemistry and model systems have brought much progress towards the understanding of human infectious, hereditary and somatically acquired diseases. The knowledge of carcinogenesis has increased very rapidly in the past few years, particularly with the development of automated molecular biologic analysis of tumors and preneoplastic lesions. Despite the wide variety of studies on the potential use of telomerase as a cancer biomarker, the variability of reported telomerase activity and the lack of a transferable detection method have prevented it from becoming a routine clinical application. Real-time PCR is a clinically transferable method and the advancement of real-time measurements of telomerase will facilitate moving telomerase activity and technologies towards clinical validation. It is expected that the next 5 years will see telomerase integrated into the initial detection and follow-up monitoring of cancer patients. The hope is that the use of telomerase will finally translate into a diagnostic to help realize longer survival and a better quality of life.     

视网膜母细胞瘤中DLC-1启动子甲基化异常

目的探讨肝癌缺失基因1(deleted in liver cancer-1,DLC-1)启动子在视网膜母细胞瘤(retinoblastoma,RB)中的甲基化情况及与临床参数的关系。方法收集37例RB石蜡切片,4例正常眼部组织标本和2株RB细胞系(WERI-Rb1及Y79)。用甲基化敏感性高分辨率熔解(MS-HRM)法定量检测DLC-1甲基化情况,用亚硫酸氢盐测序对其进行验证,并用RT-PCR检测WERI-Rb1和Y79细胞系中DLC-1 mRNA的表达。结果 35.1%的RB中存在DLC-1启动子甲基化,而在正常眼部组织中未发现甲基化;WERI-Rb1和Y79检测出DLC-1 mRNA表达。DLC-1启动子甲基化与肿瘤分化、伴随其他肿瘤和家族史均无显著关系,但单侧RB患者中的DLC-1甲基化率明显高于双侧患者(P<0.05)。结论 RB患者DLC-1启动子存在一定程度的甲基化,DLC-1甲基化在RB的发生、发展中具有一定的作用。     

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.     

The potential use of cell-free-circulating-tumor DNA as a biomarker for prostate cancer

Introduction: We are yet to identify an accurate, precise and non-invasive biomarker for the detection of prostate cancer. It would undoubtedly be useful to have a reliable and cost-effective biomarker to inform clinical practice, in order to make a non-invasive diagnosis and to predict risk of progression to aggressive prostate cancer. Since the detection of cell-free-circulating-tumor DNA in the body fluids of prostate cancer patients, a number of studies have been conducted to assess diagnostic and/or prognostic information.

Areas covered: In this literature review we evaluate the utility of cell-free-circulating-tumor-DNA for the development of a diagnostic and/or prognostic tool for prostate cancer. In addition, we identify potential areas for future research. Results from both quantitative and qualitative studies are presented.

Expert commentary: Evidence for the suitability of a panel of DNA methylation markers for the non-invasive diagnosis of prostate cancer is strong. This panel would likely include the assessment of methylation status in gene promoter regions within the EDNR, GSTP1 and MDR genes. TIMP3 and APC show potential as diagnostic markers and should be further researched. Similarly, quantitation of cell-free-circulating-tumor-DNA in blood and urine requires further investigation.     

端粒酶的结构功能与前列腺癌的相关性研究进展

目的:端粒酶是一种特殊的DNA聚合酶,它能以自身内源性RNA为模板合成端粒序列并添加到染色体末端上,稳定染色体,阻止细胞分裂衰亡。端粒酶的激活是肿瘤获得无限生长能力的关键步骤,是众多肿瘤发展所必需的。对端粒酶的结构、功能及其对前列腺癌早期诊断、预防及为保存前列腺器官功能的早期治疗中的价值进行综述。资料来源:应用计算机检索Medline数据库1990-01/2003-12相关端粒酶的文章,检索词“telomerase,prostateneoplasm,限定文章语言种类为英文。同时计算机检索中国期刊全文数据库、万方数据库2000-01/2003-12相关端粒酶的文章,检索词“端粒酶;前列腺肿瘤”,限定文章语言种类为中文。资料选择:对资料进行初审。纳入标准:有关端粒酶的结构功能与前列腺癌相关性的文献。排除标准:文献中重复研究、综述、Meta分析类文章。资料提炼:共收集到13篇有关端粒酶结构功能的文献,10篇有关端粒酶与前列腺癌相关性的文献。查找全文。资料综合:将所选资料按照以下顺序归纳总结:①端粒酶的结构与功能。②端粒酶与肿瘤的关系。③端粒酶在前列腺癌诊断、治疗中的应用。结论:端粒酶的激活与细胞的永生化和恶性肿瘤的发生发展密切相关。端粒酶活性的检测和以端粒酶为靶点的基因治疗为前列腺癌预防和早期干预提供实验学价     

血清 GSTP1基因甲基化定量分析在肝细胞癌的早期诊断价值

目的：建立实时荧光定量甲基化方法研究肝细胞癌（HCC）患者外周血血清中游离谷胱甘肽-硫-转移酶P1基因（GSTP1）启动子区域甲基化状态,探讨其是否可作为HCC早期诊断的指标。方法收集95例血清标本,包括40例HCC、30例肝硬化和25例健康体检者,采用实时荧光定量甲基化特异性聚合酶链反应（PCR）技术对血清中GSTP1基因的甲基化水平进行定量分析,应用接受者操作特性（ROC）曲线评估其诊断价值。结果肝癌患者血清中GSTP1基因甲基化定量水平明显高于健康对照组,差异有统计学意义（P＜0．05）。ROC曲线分析表明通过GSTP1基因甲基化定量分析能够高效区分肝癌和肝硬化及健康者（AUC＝0．8641）,以甲基化率2％作为诊断HCC的临界值,其诊断特异度为87．5％,敏感度达69．6％;联合检测血清GSTP1基因甲基化和血清AFP可将HCC检出率提高至75％。结论实时荧光定量甲基化方法可精确定量血清中GSTP1基因甲基化水平,在HCC的早期诊断中有一定的应用价值。     

Prostate-specific antigen in screening of prostate cancer

Prostate-specific antigen (PSA) is a wellcharacterized human prostate-specific glycoprotein. PSA has been shown to be the most effective immunohistologic marker for prostate cancer, as well as the most useful serologic test in staging and monitoring prostate cancer and in early detection of recurrent disease. The greatest clinical value of PSA is as an aid for early detection of prostate cancer. Recent studies have indicated that PSA-based screening of the older population for organ-confined early-stage prostate cancer is an acceptable, practical, and reliable modality. The accuracy of PSA screening is within the same range as the mammogram. The cost-effectiveness of PSA is comparable to other cancer screening tests. Although the increase in the patient's survival due to PSA-based detection of early prostate cancer remains to be documented, it is generally agreed that the PSA test along with digital rectal examination (DRE) should be included in the annual physical examination for men 50 years of age or older. Highrisk men are urged to commence at age 40. Asymptomatic men who have both a negative DRE and normal PSA blood test need only to continue an annual DRE and PSA check-up. Men who have a negative DRE and elevated PSA, and all those who have a suspicious DRE regardless of PSA results, should undergo further diagnostic workup, such as transrectal ultrasonography with biopsy of visible lesions. The cure rate is high with timely treatment, when prostate cancer is detected while still confined to the prostate. © 1994 Wiley-Liss, Inc.     

Urine markers as possible tools for prostate cancer screening: review of performance characteristics and practicality

BACKGROUND: In recent years, an increasing number of urine-based tests have been proposed as potential screening tests for prostate cancer. The goal of this review was to summarize the current status of evidence regarding performance characteristics of the proposed tests and their practicality under screening conditions. METHOD: Relevant articles published up to and including May 2005 were identified in the PubMed database. At least 10 cases and 10 controls had to be analyzed for a study to be included in the review. Data concerning the study population, performance characteristics, and the collection and processing of urine samples were extracted from the reviewed articles. RESULTS: In all, 34 retrospective studies evaluating 21 different markers complied with the inclusion criteria. Most of the studies were rather small and included heterogeneous clinical study populations. Promising results were reported for a few markers in single studies, but they have often not been replicated in subsequent larger studies. Some of the more promising results were obtained with 24-h urines or with specimen-handling procedures that might be difficult to perform under screening conditions. CONCLUSIONS: Larger studies with a prospective design are required to confirm promising findings regarding performance characteristics of some novel markers recently reported in mostly small studies. Future studies should also pay particular attention to the practicality of the markers under screening conditions.     

DLC-1基因启动子甲基化对于B-ALL的预后价值

目的观察儿童急性淋巴细胞白血病中肝癌缺失-1（DLC-1）抑癌基因启动子甲基化状态,评估该基因甲基化对于急性B淋巴细胞白血病（B-ALL）的预后价值。方法对40例B-ALL患儿DLC-1启动子甲基化状态与白血病敏感预后指标白血病微小残留病灶（MRD）,以及其他对B-ALL预后可能有影响的指标作一比较,评价DLC-1对于B-ALL的预后判断价值,以及是否可以作为B-ALL的独立预后危险因素。结果 DLC-1甲基化阳性的B-ALL患儿的复发率和5年无事件生存率均与DLC-1甲基化阴性患儿差异有统计学意义（P〈0.05）。DLC-1启动子甲基化与MRD的结果分布并不一致（P〉0.05）。但两者同为阳性结果的5例患儿在5年内全都复发,复发率为100%;而两者同为阴性结果的11例患儿,仅1例在5年内复发,复发率为9%。多因素分析显示仅MRD可作为独立的预后危险因素（P=0.017）,DLC-1基因甲基化阳性患儿复发风险是甲基化阴性患儿的8.5倍。结论 DLC-1甲基化和MRD都是B-ALL有意义的预后指标,两者结合可为临床提供覆盖面更广的预后信息。DLC-1甲基化也显示了该指标有成为独立预后危险因素的趋势。