循环DNA和HBV—DNA载量与原发性肝癌的相关性探讨

目的：探讨血浆DNA和HBV—DNA载量在病毒性肝炎肝硬化后原发性肝癌（PHC）的诊断价值。方法：用RT—PCR方法随访检测HBsAg阳性且经彩色B超诊断为肝硬化的患者,每6个月检测一次患者的血液循环DNA和HBV-DNA载量,直到经临床症状、彩色B超、CT诊断为原发性肝癌为止。结果：在PHC中,循环DNA61．3±31．9ng／ml,健康对照组20．3±9．9ng／ml,两组差异显著（P〈0．01）;而HBV—DNA载量,在PHC确诊时多数处于较低水平,78％≤105copies／ml,与PHC确诊前有明显差异（P〈0．01）。PHC未发生淋巴结转移者循环DNA51．2±17．9ng／ml,淋巴结转移者循环DNA73．9±23．7ng／ml,两者存在差异（P〈0．05）;而HBV-DNA载量有无淋巴结转移无明显差异。结论：联合检测血液循环DNA和HBV—DNA载量对PHC的预测、预后价值明显,且方法简单,易于开展。     

HBV阳性原发性肝癌的高危因素探讨

目的探讨HBV阳性原发性肝癌（HCC）与血清HBV-DNA水平、饮酒史及性别因素的关系。方法回顾性分析浙江大学医学院附属第一医院2005年1月至2007年7月住院的HBV阳性HCC401例和同期住院的慢乙肝（CHB）患者401例,采用以年龄为配比变量的1：1匹配的病例对照研究,将HBsAg、HBeAg、HBcAb阳性分为大三阳组,HBsAg、HBeAb、HBcAb阳性分为小三阳组。结果计量资料采用配对t检验,计数资料采用Pearson卡方检验,结果提示不管是大三阳组还是小三阳组,HBV DNA水平、饮酒史和性别均与HCC有关系;Logistic回归分析HBV DNA、饮酒史和性别与HCC的关系,同样提示HBV DNA水平、饮酒史和性别与HCC的发病有关系。结论HBV DNA水平、性别和饮酒史在HBV阳性的原发性肝癌的发生中占重要作用,对HBV DNA高水平的男性饮酒患者,更应建议戒酒并采取合适的抗病毒治疗方案,并加强监测随访,降低肝癌的发生率或及早发现肝癌。     

原发性肝癌ATM基因启动子甲基化及其与放疗疗效的相关性

为探究原发性肝癌(hepatocellular carcinoma, HCC)共济失调-毛细血管扩张突变基因(ataxia-telangiectasia mutated gene, ATM)启动子甲基化情况,分析其与临床特征的关系及与放疗疗效的相关性,采用甲基化特异性PCR法(methylation-specific PCR, MSP)检测50对肝癌手术标本及相应的癌旁肝组织标本、20例正常肝组织、38例局部中晚期肝癌肝穿刺标本的ATM基因启动子甲基化状态,并采用BSP (bisulfite sequencing PCR)测序法对样本的甲基化状态进行验证,分析ATM基因启动子甲基化状态与患者临床特征的关系及与放疗疗效的相关性。结果发现, 88例肝癌组织中ATM基因启动子甲基化率为39.8%(35/88),其中38例肝癌肝穿刺标本有42.1%(16/38)存在ATM基因启动子甲基化, 50例肝癌手术标本有38%(19/50)存在ATM基因启动子甲基化,相应癌旁肝组织只有8.0%(4/50)存在ATM基因启动子甲基化;正常肝组织未发现有ATM基因启动子甲基化, ATM基因启动子甲基化在肝癌组织中的发生率与癌旁肝组织、正常肝组织相比差异具有统计学意义(χ2=24.818,P0.05)。此外,存在ATM基因启动子甲基化的局部中晚期肝癌患者的放疗疗效显著优于无甲基化的病例(χ2=5.955,P=0.015), ATM基因启动子甲基化状态与肝癌放疗疗效呈显著正相关关系(r=0.396, P=0.014)。实验结果表明原发性肝癌ATM基因启动子存在异常甲基化, ATM基因启动子甲基化状态与肝癌放疗疗效关系密切,可为筛选放射敏感差异病人提供新的思路。     

原发性肝癌临床分期的对比探讨

原发性肝癌严重威胁着人类的健康,全球每年新增的病例数约为50万,其中54％发生在我国,且患者在3年内复发率高于60％,可见诊治形势十分严峻。肝癌的临床分期系统对于患者的预后评估以及选择何种治疗方案有着极其重要的意义。国内外虽有此类研究的相关内容,但将各种分期的评价及适用范围、发展趋势相结合进行分析的较少。本文对国际上多种常用的分期方案（TNM分期、Child-Pu曲分期、Okuda分期、CLIP评分、JIS分期、CUPI指标、CIS记分）进行探讨,论述各种方案间的差异及相互关系,并对未来可能发展方向进行展望。经本文分析多项研究发现：TNM分期更适用于外科病人,Child—Pugh对肝功能受损的患者具有较好的预后价值,而Okuda、CLIP、JIs分期适用于不适合手术治疗的进展期病人,CUPI指数则对慢性乙肝患者有疗效,CIS适用于非手术治疗的患者。随着肿瘤分子生物学技术的进步,肝癌分期有可能上升到分子病理学的水平,肝癌切除的根治程度将划分得更为细致,以反映患者肝癌切除后所处状态的多种可能性,分子指标也将更加客观、敏感,有利于患者早期的诊断与治疗。     

原发性肝癌的介入治疗现状与进展

原发性肝癌(Hepatocellular carcinoma,HCC)是肝脏最常见的恶性肿瘤,具有高发病率、死亡率及预后差的特点,大多数患者确诊时为进展期,已失去手术机会,严重危及患者生命。虽然目前相关的治疗方法较多,但预后仍较差,介入治疗作为一种微创疗法在提高患者生存期、改善患者生活质量等方面取得显著效果。本文就原发性肝癌的介入治疗现状与进展进行综述。     

乙肝病毒与原发性肝癌的相关风险研究

为了解慢性乙型肝炎病毒感染与原发性肝癌的关系,本文采用回顾性研究方法对 328 例原发性肝癌病人与同期收治的 340 例非肝癌的其他消化道肿瘤病人的乙型肝炎病毒感染血清标志物(HBV M)及肝功能检测结果进行对比分析。结果显示肝癌组乙肝表面抗原(HBsAg)阳性率(63.11%)显著高于非肝癌组(消化道其他肿瘤对照组)(11.47%)。肝癌组慢性乙型肝炎病毒感染“HBsAg、抗-HBe 和抗-HBc 三者均表达为阳性者”(37.2%)显著高于“HBsAg、HBeAg 和抗-HBc 三者均表达为阳性者”(6.4%)。肝功能检测结果,“HBsAg、HBeAg 和抗-HBc三者均表达为阳性组”与“HBsAg、HBeAg 和抗-HBc 三者均表达为阳性组”比较无显著性差异(P>0.05),而肝癌组与非肝癌组比较,肝癌组肝损害显著高于非肝癌组(P<0.01)。表明慢性乙型肝炎病毒感染在原发性肝癌病因学中起着十分重要的作用,“HBsAg、抗-HBe 和抗-HBc 三者均表达为阳性者”是原发性肝癌的高危人群。     

超声诊断原发性肝癌的研究

本文33例原发性肝癌的超声诊断研究中24例有1.5至30年的肝病史。最近一次确诊为肝硬化者或无肿瘤者到首次发现肿瘤的时间为3至98天,平均27.3天,其中一例仅隔3天再查就发现肝左叶有多发性低回声肿瘤,最大肿瘤为64×65mm;首次发现肿瘤到第二次复查的时间为15至158天,平均49.4天,肿瘤平均增大约1.5倍,从单个发展至多个。本文就上述原发性肝癌发展的超声诊断跟踪分析探讨。     

肝癌组织中线粒体DNA D-Loop区碱基变异与ROS水平

为了探讨ROS水平与突变的关系,对原发性肝癌线粒体DNA区的突变情况进行研究,同时对原发性肝癌患者组织细胞内ROS进行测定。选择20例原发性肝癌组织及其邻近的癌旁组织,用PCR方法将线粒体DNA D-Loop扩增后测序。组织内ROS的水平采用流式细胞技术测定。结果表明在20对原发性肝癌组织中存在8对mtDNA突变,突变率为40%,共发现突变位点53个,包括2个插入,11个缺失,40个点突变,其中T-C,C-T的转换占75%,4个属于微卫星结构。癌组织突变一般伴有癌旁组织突变,癌组织突变位点高于癌旁组织。发现一例标本的癌组织和癌旁组织均有大片段丢失。原发性肝癌组织内ROS水平明显高于癌旁对照( P<0.01),同时我们发现在区发生突变的患者的组织中ROS水平明显高于未发生突变的肝癌组织标本（P<0.01）,发生突变的癌旁组织内ROS水平明显高于未发生突变的癌旁组织（P<0.01）。结论 (1)线粒体DNA D-Loop区是一个高度多态性和突变性的区域,在原发性肝癌中突变率较高。(2)肝癌患者组织细胞内ROS异常,提示肝癌的线粒体DNA发生的点突变及肝癌的发生可能与ROS升高有关。     

原发性肝癌的表观遗传学及其治疗

肝癌是一种严重危害人类健康的恶性疾病,在全世界患癌人群中,肝癌的发生率排第五,死亡率排第二。原发性肝癌(Hepatocellular carcinoma, HCC)是最普遍的肝癌组织学亚型,属于异质性疾病,对其治疗涉及遗传学、基因组学、环境毒理学等多个领域。尽管许多分子靶向治疗药物如索拉菲尼等已经进入临床应用并证明有效,但细胞毒性等负效应不容忽视,目前迫切需要新的治疗靶点和药物高效并选择性的杀伤肝癌细胞。大量证据表明,肝脏肿瘤的发生和发展与表观遗传学密切相关,DNA甲基化、组蛋白修饰、miRNA表达的异常及表观遗传相关基因表达的异常都是HCC中显著的表观遗传异常现象。表观治疗药物可能会逆转异常基因的表达,从而使HCC的发生和发展得以控制。文章综述了HCC表观遗传学治疗方面的研究进展,展望了未来利用类似的疗法治疗肝癌的潜力。     

乙肝病毒与原发性肝癌的相关风险研究

为了解慢性乙型肝炎病毒感染与原发性肝癌的关系,本文采用回顾性研究方法对328例原发性肝癌病人与同期收治的340例非肝癌的其他消化道肿瘤病人的乙型肝炎病毒感染血清标志物(HBV M)及肝功能检测结果进行对比分析.结果显示肝癌组乙肝表面抗原(HBsAg)阳性率(63.11%)显著高于非肝癌组(消化道其他肿瘤对照组)(11.47%).肝癌组慢性乙型肝炎病毒感染"HBsAg、抗-HBe和抗-HBc三者均表达为阳性者"(37.2%)显著高于"HBsAg、HBeAg和抗-HBc三者均表达为阳性者"(6.4%).肝功能检测结果,"HBsAg、HBeAg和抗-HBc三者均表达为阳性组"与"HBsAg、HBeAg和抗-HBc三者均表达为阳性组"比较无显著性差异(P＞0.05),而肝癌组与非肝癌组比较,肝癌组肝损害显著高于非肝癌组(P＜0.01).表明慢性乙型肝炎病毒感染在原发性肝癌病因学中起着十分重要的作用,"HBsAg、抗-HBe和抗-HBc三者均表达为阳性者"是原发性肝癌的高危人群.     

血浆循环DNA水平与血常规指标的关联分析

目的:通过观察血浆循环DNA(cfDNA)浓度与血常规指标间的关系,探索cfDNA的来源。方法:用实时荧光定量PCR测定选定人群的血浆cfDNA浓度,用ADVIA120血细胞分析仪测定血常规指标,用双变量关联分析和逻辑回归分析血浆cfDNA浓度与性别、年龄及血常规指标间的相关性。结果:男性的血浆cfDNA浓度(23.23±3.186)略高于女性(18.55±2.037),但差异无显著性(P=0.2183);在各年龄段中,61~70岁年龄段的cfDNA浓度(23.20±4.274)最高,其次为70岁以上年龄段(20.92±2.089),51~60岁年龄段最低(15.81±1.747),但三者之间的差异没有显著性(P=0.3276);双变量关联分析表明血浆cfDNA浓度与过氧化物酶活性指数、血红蛋白及红细胞比积呈正相关(P<0.05),与性别、年龄及其他血常规指标无关;逻辑回归分析提示血浆cfDNA浓度升高只与平均血小板浓度和过氧化物酶活性指数呈正相关(P<0.05)。结论:血浆cfDNA增加可能与平均血小板浓度升高及过氧化物酶含量或活性有关,血小板前体细胞(即巨核细胞)可能是cfDNA的一个重要来源。     

循环肿瘤DNA的检测技术及在癌症诊疗中的应用价值

循环肿瘤DNA（ctDNA）来源于肿瘤,能很好地反映肿瘤的基因信息,并且会随着肿瘤的进展发生相应的改变。近年来,ctDNA独特的能力备受人们关注并被广泛研究,本文在总结ctDNA的来源、性质和样品处理的基础上,对ctDNA的检测技术及在癌症诊疗中的应用进行综述,阐述了ctDNA标准物质在二代测序中的作用及重要性,并指出ctDNA样品的收集、存储、定量检测以及数据分析等各个流程中建立统一标准及规范的迫切性。     

HBV—DNA的生物素寡聚核苷酸探针快速检测

本文建立一种快速ＨＢＶ－ＤＮＡ检测方法,此法是基于生物素标记的寡聚核苷酸与硝酸纤维素滤膜上的靶ＤＮＡ杂交,然后通过Ｓｔｒｅｐｔｏａｖｉｄｉｎ－碱性磷酸酶偶联体及生色底物进行检测。本文研究了此法的实用性,表明对于实验室及临床的ＨＢＶ－ＤＮＡ检测,此法是一种快速（４小时）、灵敏（１－１０ｐｇ）、特异、方便的方法。     

HBV-DNA 与乙型肝炎血清标志物的相关性分析

目的：探讨乙型肝炎病毒(HBV)DNA载量与其血清标志物的相关性。方法：运用荧光定量聚合酶链反应(FQ-PCR)、酶联免疫 吸附实验(ELISA)分别检测503 例患者HBV-DNA 载量和HBV 血清标志物。根据HBV 血清标志物结果分为大三阳组、小三阳 组、少见模式组、抗体阳性及全阴组,比较各组间HBV-DNA的阳性率及定量值。结果：在大三阳组、小三阳组、少见模式组、抗体 阳性及全阴组HBV-DNA 的阳性率分别为90%、65.1%、65.2%、2.0%,HBV-DNA 的定量结果(logHBV-DNA) 别为6.32± 1.96、 2.01± 1.68、3.48± 2.52 (抗体阳性及全阴组阳性例数过低,不纳入统计)。大三阳组HBV-DNA的阳性率显著高于小三阳组(P<0. 05),大三阳组、小三阳组HBV-DNA 的阳性率与少见模式组比较,差异均无统计学意义(P>0.05),但大三阳组、小三阳组、少见模式 组HBV-DNA 的阳性率均显著高于抗体阳性及全阴组(P<0.01)。HBsAg、HBeAg 阳性组HBV-DNA 的阳性率分别显著高于HBsAg 、HBeAg 阴性组(P<0.01)。小三阳组、少见模式组HBV-DNA 载量均显著低于大三阳组(P<0.01),少见模式组HBV-DNA 载量 显著高于小三阳组(P<0.05)。结论：HBV-DNA的阳性率与HBeAg、HBsAg相关;HBV-DNA载量与HBV 血清标志物模式相关。     

Methylation-capture and Next-Generation Sequencing of free circulating DNA from human plasma

Background

Free circulating DNA (fcDNA) has many potential clinical applications, due to the non-invasive way in which it is collected. However, because of the low concentration of fcDNA in blood, genome-wide analysis carries many technical challenges that must be overcome before fcDNA studies can reach their full potential. There are currently no definitive standards for fcDNA collection, processing and whole-genome sequencing. We report novel detailed methodology for the capture of high-quality methylated fcDNA, library preparation and downstream genome-wide Next-Generation Sequencing. We also describe the effects of sample storage, processing and scaling on fcDNA recovery and quality.

Results

Use of serum versus plasma, and storage of blood prior to separation resulted in genomic DNA contamination, likely due to leukocyte lysis. Methylated fcDNA fragments were isolated from 5 donors using a methyl-binding protein-based protocol and appear as a discrete band of ~180 bases. This discrete band allows minimal sample loss at the size restriction step in library preparation for Next-Generation Sequencing, allowing for high-quality sequencing from minimal amounts of fcDNA. Following sequencing, we obtained 37×10⁶-86×10⁶ unique mappable reads, representing more than 50% of total mappable reads. The methylation status of 9 genomic regions as determined by DNA capture and sequencing was independently validated by clonal bisulphite sequencing.

Conclusions

Our optimized methods provide high-quality methylated fcDNA suitable for whole-genome sequencing, and allow good library complexity and accurate sequencing, despite using less than half of the recommended minimum input DNA.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-476) contains supplementary material, which is available to authorized users.     

HBV-DNA与乙型肝炎血清标志物的相关性分析

目的：探讨乙型肝炎病毒（HBV）DNA载量与其血清标志物的相关性。方法：运用荧光定量聚合酶链反应（FQ-PCR）、酶联免疫吸附实验（ELISA）分别检测503例患者HBV．DNA载量和HBV血清标志物。根据HBV血清标志物结果分为大三阳组、小三阳组、少见模式组、抗体阳性及全阴组,比较各组间HBV—DNA的阳性率及定量值。结果：在大三阳组、小三阳组、少见模式组、抗体阳性及全阴组HBV—DNA的阳性率分别为90％、65．1％、65．2％、2．0％,HBV—DNA的定量结果（10gHBV—DNA）别为6．32±1．96、2．01±1．68、3．48＋2．52f抗体阳性及全阴组阳性例数过低,不纳入统计）。大三阳组HBV—DNA的阳性率显著高于小三阳组（P〈0．05）。大三阳组、小三阳组HBV—DNA的阳性率与少见模式组比较,差异均无统计学意义（P〉0．05）,但大三阳组、小三阳组、少见模式组HBV．DNA的阳性率均显著高于抗体阳性及全阴组（P〈0．01）。HBsAg、HBeAg阳性组HBV—DNA的阳性率分别显著高于HB—sAg、HBeAg阴性组（P〈0．01）。小三阳组、少见模式组HBV．DNA载量均显著低于大三阳组（P〈0．01）,少见模式组HBV—DNA载量显著高于小三阳组（P〈0．05）。结论：HBV—DNA的阳性率与HBeAg、HBsAg相关;HBV—DNA栽量与HBV血清标志物模式相关。     

Circulating DNA in the blood and its application in medical diagnosis

Circulating nucleic acids were discovered more than 30 years ago, but did not attract much attention until the past decade. This review summarizes the data on the sources of extracellular DNA circulating in the blood, features of its circulation, and pathways of its removal. The possibility of using circulating DNA in medical diagnosis is discussed.     

Circulating Tumor DNA as Biomarkers for Cancer Detection

Detection of circulating tumor DNAs (ctDNAs) in cancer patients is an important component of cancer precision medicine ctDNAs. Compared to the traditional physical and biochemical methods, blood-based ctDNA detection offers a non-invasive and easily accessible way for cancer diagnosis, prognostic determination, and guidance for treatment. While studies on this topic are currently underway, clinical translation of ctDNA detection in various types of cancers has been attracting much attention, due to the great potential of ctDNA as blood-based biomarkers for early diagnosis and treatment of cancers. ctDNAs are detected and tracked primarily based on tumor-related genetic and epigenetic alterations. In this article, we reviewed the available studies on ctDNA detection and described the representative methods. We also discussed the current understanding of ctDNAs in cancer patients and their availability as potential biomarkers for clinical purposes. Considering the progress made and challenges involved in accurate detection of specific cell-free nucleic acids, ctDNAs hold promise to serve as biomarkers for cancer patients, and further validation is needed prior to their broad clinical use.     

Mutational Analysis of Circulating Tumor Cells from Colorectal Cancer Patients and Correlation with Primary Tumor Tissue

Circulating tumor cells (CTCs) provide a non-invasive accessible source of tumor material from patients with cancer. The cellular heterogeneity within CTC populations is of great clinical importance regarding the increasing number of adjuvant treatment options for patients with metastatic carcinomas, in order to eliminate residual disease. Moreover, the molecular profiling of these rare cells might lead to insight on disease progression and therapeutic strategies than simple CTCs counting. In the present study we investigated the feasibility to detect KRAS, BRAF, CD133 and Plastin3 (PLS3) mutations in an enriched CTCs cell suspension from patients with colorectal cancer, with the hypothesis that these genes` mutations are of great importance regarding the generation of CTCs subpopulations. Subsequently, we compared CTCs mutational status with that of the corresponding primary tumor, in order to access the possibility of tumor cells characterization without biopsy. CTCs were detected and isolated from blood drawn from 52 colorectal cancer (CRC) patients using a quantum-dot-labelled magnetic immunoassay method. Mutations were detected by PCR-RFLP or allele-specific PCR and confirmed by direct sequencing. In 52 patients, discordance between primary tumor and CTCs was 5.77% for KRAS, 3.85% for BRAF, 11.54% for CD133 rs3130, 7.69% for CD133 rs2286455 and 11.54% for PLS3 rs6643869 mutations. Our results support that DNA mutational analysis of CTCs may enable non-invasive, specific biomarker diagnostics and expand the scope of personalized medicine for cancer patients.