肿瘤病人外周血DNA检测的意义

肿瘤病人血循环中DNA的量较一般人明显增多,而且从中可检测到肿瘤相关基因的特征性改变,可用于协助诊断,并作为预测预后及疗效观察的指标,具有重要的临床意义。     

游离DNA检测对肿瘤诊断的意义

肿瘤患者血液、胸腹腔积液、脑脊液等体液中可以检测到游离DNA含量远大于正常人,并且游离DNA表现出与肿瘤组织相同的生物学特性.本文综述游离DNA的部分肿瘤特性,主要是游离DNA甲基化、基因突变和微卫星改变等,介绍游离DNA检测对肿瘤患者的诊断、预后跟判断复发等方面的作用.     

血清肿瘤标志物联合检测在恶性肿瘤诊断中的临床应用

目的　检测恶性肿瘤血清中癌相关抗原 (CA19- 9、CA12 5、CA15 - 3) ,以及恶性肿瘤相关物质群(TSGF)。评价其对恶性肿瘤的诊断、病情监测和预后判断的价值。方法　检测 316例恶性肿瘤及 30例健康人血清中CA19- 9、CA12 5、CA15 - 3及TSGF的含量。结果　TSGF在恶性肿瘤组中含量明显高于正常对照组 ,其差异有显著性 (P <0 0 1) ,敏感性为 80 4 % ,特异性达 96 7% ,准确性为 81 8%。恶性肿瘤组中CA19- 9、CA12 5、CA15～ 3与正常对照组比较也有显著性差异 (P <0 0 1) ,四项联合检查的敏感性及准确性均较单项检测高 ,其中在卵巢癌组中的敏感性高达 91 3% ,其次为肺癌组 ,为 86 8%。结论　血清恶性肿瘤标志物的单项检测对某些肿瘤有显著意义 ,但联合检测对恶性肿瘤的早期诊断及病情监测和预后判断更有价值     

肝癌患者肿瘤循环DNA的检测

一、肿瘤循环DNA的发展简史早在50多年前,也就是Watson和Crick阐明DNA双螺旋结构前五年,Mandel和Me'tais在人体血浆中发现了核酸[1].1975年,Leon[2]发现肿瘤患者比正常健康人血液中有更多的循环DNA.随着PCR方法在循环DNA领域的应用,人们在胰腺癌及黑色素淋巴瘤患者血浆DNA中,首次检测到N-ras及K-ras基因突变[3,4].     

肿瘤患者外周血游离DNA的研究进展

肿瘤患者外周血中存在着游离DNA,其含量明显高出健康人水平,并且这些游离DNA具有肿瘤特征性。定量这些游离DNA并分析其肿瘤特征性(主要介绍微卫星的改变,如杂合性缺失LOH及微卫星不稳定性MSI),对肿瘤的诊断、治疗及预后的评价都具有重要意义。     

外周血DNA及其遗传学改变与肿瘤转移复发

随着近年来肿瘤细胞生物学及分子生物学的深入研究，以及分子生物学检测技术的快速发展，外周血肿瘤标记由于检测的方便和非侵入性已逐步替代了受到标本采集以及无法连续检测和随访追踪等诸多限制的组织学肿瘤标记。外周血DNA及其改变的分子遗传学检测正以其不可替代的优点逐渐被人们所重视，并成为肿瘤细胞生物学及分子生物学研究中引人注目的一个亮点。     

外周血白细胞DNA甲基化与肿瘤发病相关性Meta分析

目的通过Meta分析探讨外周血DNA甲基化与癌症风险相关性。方法分别在4个英文数据库（PubMed,ISI WOK databases,Science Online,OVID）和3个中文数据库（CNKI,万方和VIP）中系统检索文献,检索日期截至2013-08,英文数据库检索的主题词为DNA methylation,blood or leukocyte和cancer,tumor,carcinoma,neoplasm or malignancy,不限制语言,中文数据库检索的主题词为DNA甲基化,外周血或白细胞和肿瘤或癌。文献质量评估应用Newcastle-Ottawa Scale（NOS）。应用固定效应模型（异质性检验I2〈25%）或随机效应模型（异质性检验I2≥25%）估计总体比值比（OR）和95%可信区间（95%CI）。结果共有22项外周血全基因组DNA甲基化和20项外周血特定基因DNA甲基化研究纳入分析,外周血全基因组DNA低甲基化（OR=1.34,95%CI：1.12-1.60,I2=89.1%,P=0.001）和外周血特定位点DNA过甲基化（OR=1.37,95%CI：1.24-1.52,I2=93.2%,P〈0.001）增加肿瘤总体发病风险。肿瘤类型亚族分析发现,外周血全基因组DNA低甲基化水平增加膀胱癌（OR=1.96,95%CI：1.44-2.48,I2=85.1%,P=0.001）、结直肠癌（OR=1.85,95%CI：1.36-2.35,I2=61.1%,P=0.012）、胃癌（OR=1.38,95%CI：1.08-1.68,I2=37.7%,P=0.170）和肝细胞肝癌（OR=1.38,95%CI：1.03-1.73,I2=77.3%,P=0.012）的发病风险。结论外周血DNA甲基化水平与肿瘤发生存在相关性,可能成为流行病学人群筛查的重要生物指标。     

淋巴结阴性的乳腺癌DNA含量检测的意义

淋巴结转移与否常作为判断治疗后预后的主要因素,然而在淋巴结阴性的病人中亦有部份病人出现复发或远处转移。对这些病人如果手术后选用辅助治疗可能提高其生存率,因而如何在病理上淋巴结没有转移的病人中发现哪些具有高危险复发因素的病人,是提高疗效的关键。本文应用FCM检测55例淋巴结无转移的乳腺癌患者的原发病灶的DNA含量,并同时测定其腋淋巴结的DNA含量和原发病灶的激素受体ER和PgR状态,通过30月的随访资料,以评价DNA含量的测定在淋巴结无转移乳腺癌患者的价值。     

DNA甲基化的测定及其在肿瘤诊断中的意义

ＤＮＡ甲基化的测定及其在肿瘤诊断中的意义房静远，江绍基上海第二医科大学仁济医院上海市消化疾病研究所（上海２００００１）真核细胞在转录水平的基因调控作用之一，是ＤＮＡ特殊部位的甲基修饰作用。ＤＮＡ甲基化（ＤＮＡｍｅｔｈｙｌａｔｉｏｎ）状态的维持与基因转...     

乳腺癌患者血清游离DNA的定量检测

目的:定量检测乳腺癌患者血清中的游离DNA,并评估其应用前景。方法:采用荧光定量PCR方法检测100例健康女性志愿者、100例乳腺良性疾病患者和200例乳腺癌患者血清游离DNA中GAPDH基因拷贝数,以此作为评估DNA含量的指标。结果:以≥1×103基因拷贝数作为阳性标准,93%健康女性和95%乳腺良性疾病患者DNA检测为阴性,而84.5%乳腺癌患者DNA检测为阳性,其中Ⅰ～Ⅱ期乳腺癌患者阳性率为84%。DNA含量在3组之间的差异有统计学意义(P<0.005)。结论:早期乳腺癌患者血清中存在一定数量肿瘤源性的游离DNA,此类遗传物质可以作为一类特异性的生物学标志物。     

Clinical implications of plasma circulating tumor DNA in gynecologic cancer patients

Molecular characterization of cancers is important in dictating prognostic factors and directing therapy. Next‐generation sequencing of plasma circulating tumor DNA (ctDNA) offers less invasive, more convenient collection, and a more real‐time representation of a tumor and its molecular heterogeneity than tissue. However, little is known about the clinical implications of ctDNA assessment in gynecologic cancer. We describe the molecular landscape identified on ctDNA, ctDNA concordance with tissue‐based analysis, and factors associated with overall survival (OS) in gynecologic cancer patients with ctDNA analysis. We reviewed clinicopathologic and genomic information for 105 consecutive gynecologic cancer patients with ctDNA analysis, including 78 with tissue‐based sequencing, enrolled in the Profile‐Related Evidence Determining Individualized Cancer Therapy ({"type":"clinical-trial","attrs":{"text":"NCT02478931","term_id":"NCT02478931"}}NCT02478931) trial at the University of California San Diego Moores Cancer Center starting July 2014. Tumors included ovarian (47.6%), uterine (35.2%), cervical (12.4%), vulvovaginal (2.9%), and unknown gynecologic primary (1.9%). Most ovarian and uterine cancers (86%) were high grade. 34% (N = 17) of ovarian cancers had BRCA alterations, and 22% (N = 11) were platinum sensitive. Patients received median 2 (range 0–13) lines of therapy prior to ctDNA collection. Most (75.2%) had at least one characterized alteration on ctDNA analysis, and the majority had unique genomic profiles on ctDNA. Most common alterations were TP53 (N = 59, 56.2% of patients), PIK3CA (N = 26, 24.8%), KRAS (N = 14, 13.3%), BRAF (N = 10, 9.5%), ERBB2 (N = 8, 7.6%), and MYC (N = 8, 7.6%). Higher ctDNA maximum mutation allele frequency was associated with worse OS [hazard ratio (HR): 1.91, P = 0.03], while therapy matched to ctDNA alterations (N = 33 patients) was independently associated with improved OS (HR: 0.34, P = 0.007) compared to unmatched therapy (N = 28 patients) in multivariate analysis. Tissue and ctDNA genomic results showed high concordance unaffected by temporal or spatial factors. This study provides evidence for the utility of ctDNA in determining outcome and individualizing cancer therapy in patients with gynecologic cancer.

Abbreviations

BMI

body mass index

BRCA

breast cancer susceptibility gene

CAP

College of American Pathologists

CI

confidence interval

CLIA

Clinical Laboratory Improvement Amendments

ctDNA

circulating tumor DNA

Del

deletion

HR

hazard ratio

ID

identification number

In/del

insertion/deletion

MAF

mutation allele frequency

NR

not reached

OS

overall survival

PREDICT

Profile‐Related Evidence Determining Individualized Cancer Therapy

SE

standard error

SNV

single nucleotide variant

UCSD

University of California San Diego

VUS

variants of unknown significance

     

Detection of mutant K-ras DNA in plasma or serum of patients with colorectal cancer.

Increased understanding of the molecular basis of colorectal cancer and recognition that extracellular DNA circulates in the plasma and serum of cancer patients enables new approaches to detection and monitoring. We used a polymerase chain reaction (PCR) assay to demonstrate mutant K-ras DNA in the plasma or serum of patients with colorectal cancer. Plasma or serum was fractionated from the blood of 31 patients with metastatic or unresected colorectal cancer and from 28 normal volunteers. DNA was extracted using either a sodium chloride or a gelatin precipitation method and then amplified in a two-stage PCR assay using selective restriction enzyme digestion to enrich for mutant K-ras DNA. Mutant K-ras DNA was detected in the plasma or serum of 12 (39%) patients, all confirmed by sequencing, but was not detected in any of the normal volunteers. K-ras mutations were detected in plasma or serum regardless of sex, primary tumour location, principal site of metastasis or proximity of chemotherapy and surgery to blood sampling. Tumour specimens available for 19 of the patients were additionally assayed for ras mutations and compared with blood specimens. Our results indicate mutant K-ras DNA is readily detectable by PCR in the plasma or serum of patients with advanced colorectal cancer. Thus, plasma- or serum-based nucleic acid amplification assays may provide a valuable method of monitoring and potentially detecting colorectal cancer.     

Detecting tumor-related alterations in plasma or serum DNA of patients diagnosed with breast cancer.

Chromosomal abnormalities are associated with the development of breast cancer, and widespread allelic loss or imbalance is frequently found in tumor tissues taken from patients with this disease. Using different markers, we studied a total of 61 patients (divided into three groups) for the presence of microsatellite instability and loss of heterozygosity (LOH) in plasma or serum DNA. Of the initial 27 patients, 35% of the tumor samples displayed LOH, whereas 15% had identical alterations in the corresponding plasma samples. In addition, the adjacent normal breast tissue of two patients also displayed LOH. In a second group of 11 patients, 45% of the tumors displayed LOH, and 27% displayed identical plasma DNA alterations; one case displayed an identical LOH in adjacent nontumor tissue. In a third series of 23 patients also studied with tetranucleotide repeats, 81% of the tumor samples displayed LOH, whereas 48% had LOH in the corresponding serum samples. The fact that small tumors (T1) of histoprognostic grade 1 or in situ carcinomas could present DNA alterations in the plasma/serum at an early stage, allied to the widely increased range of available microsatellite markers, suggests that plasma or serum DNA may become a useful diagnostic tool for early and potentially curable breast cancer.     

Genomic alterations in plasma DNA from patients with metastasized prostate cancer receiving abiraterone or enzalutamide

In patients with metastatic castrate‐resistant prostate cancer (mCRPC), circulating tumor DNA (ctDNA) analysis offers novel opportunities for the development of non‐invasive biomarkers informative of treatment response with novel agents targeting the androgen‐receptor (AR) pathway, such as abiraterone or enzalutamide. However, the relationship between ctDNA abundance, detectable somatic genomic alterations and clinical progression of mCRPC remains unexplored. Our study aimed to investigate changes in plasma DNA during disease progression and their associations with clinical variables in mCRPC patients. We analyzed ctDNA in two cohorts including 94 plasma samples from 25 treatment courses (23 patients) and 334 plasma samples from 125 patients, respectively. We conducted whole‐genome sequencing (plasma‐Seq) for genome‐wide profiling of somatic copy number alterations and targeted sequencing of 31 prostate cancer‐associated genes. The combination of plasma‐Seq with targeted AR analyses identified prostate cancer‐related genomic alterations in 16 of 25 (64%) treatment courses in the first cohort, in which we demonstrated that AR amplification does not always correlate with poor abiraterone and enzalutamide therapy outcome. As we observed a wide variability of ctDNA levels, we evaluated ctDNA levels and their association with clinical parameters and included the second, larger cohort for these analyses. Employing altogether 428 longitudinal plasma samples from 148 patients, we identified the presence of bone metastases, increased lactate dehydrogenase and prostate‐specific antigen (PSA) as having the strongest association with high ctDNA levels. In summary, ctDNA alterations are observable in the majority of patients with mCRPC and may eventually be useful to guide clinical decision‐making in this setting.     

肿瘤患者循环DNA的定量研究

目的建立纳克水平DNA定量方法,并探讨循环DNA检测在肿瘤诊断中的应用价值。方法对483例恶性肿瘤患者以微量基因组抽提试剂盒抽提血清DNA,以SYBRgreenⅠ荧光染色法行DNA定量;BRCA1(D17S579、D17S855)和p53(TP53,D17S786)微卫星位点的杂合性丢失检测采用聚合酶链反应结合聚丙烯酰胺凝胶电泳和银染的方法进行。同时与150例健康人血清的检测结果进行对照。结果SYBRgreenⅠ荧光染色可精确定量2～10ngDNA。483例恶性肿瘤患者血清DNA平均水平为(81.3±98.3)ng/ml,150例健康人血清DNA平均水平为(22.2±13.4)ng/ml,两者间差异有显著性(P<0.001);50例良性肿瘤患者血清DNA未见显著升高。在33例血清DNA含量增高的卵巢癌患者中,27例(81.8%)能测及BRCA1和p53微卫星标记中至少1个位点的杂合性丢失。结论循环DNA的定量有可能成为一种新的恶性肿瘤标志物。     

Microsatellite analysis of serum DNA in patients with head and neck cancer

We have shown previously that microsatellite alterations in serum DNA was predictive of distant metastasis in a study with 21 primary head and neck squamous cell carcinoma patients. To further investigate serum microsatellite alterations as a prognostic tool, we carried out microsatellite analysis of serum DNA with 10 markers on 152 patients with head and neck cancer. Forty-five percent (68/152) of patients had microsatellite alterations of serum DNA identical to corresponding tumor DNA. In 16 patients that had distant metastasis, 11 patients had a positive serum test (microsatellite alterations detectable in their serum DNA with one or more markers). The difference in distant metastasis rates between the negative and positive serum tests (6.0% [5/84] vs. 16.2% [11/68], RR = 2.7) was clinically significant and almost reached statistical significance (p = 0.06). When the analysis was restricted to patients with recurrent disease, a positive serum test correlated with those who developed distant metastasis (p = 0.04). Other parameters, such as development of recurrence, stage of the cancer, disease-free survival and overall survival, were not associated with a positive serum test. Detecting tumor DNA in serum by microsatellite analysis may help identify patients at risk for distant metastasis. Therefore, circulating tumor cells may contribute to the presence of tumor DNA in the serum. In the future if a serum test is positive, therapeutic approaches may by intensified, such as platinum-based chemoradiation, to reduce distant failures.     

结直肠癌患者血浆Septin9 DNA甲基化联合FOBT检测的临床应用价值

目的:探讨血浆Septin9 DNA甲基化及粪便隐血试验（FOBT）在结直肠癌（CRC）诊断中的应用价值。方法:回顾性收集2017年6月至2022年1月我院收治并经病理检查确诊的101例结直肠良性腺瘤患者、209例结直肠癌患者分别作为良性腺瘤组和结直肠癌组,选取同期在我院进行体检的98例健康人群作为正常对照组,比较三组一般资料及血浆Septin9 DNA甲基化及FOBT阳性情况,比较不同病理特征、不同临床分期结直肠癌患者血浆Septin9 DNA甲基化、FOBT阳性情况,采用受试者工作曲线（ROC）评估血浆Septin9 DNA甲基化、FOBT单项及联合检测对结直肠癌的诊断价值。结果:三组血浆Septin9 DNA甲基化、FOBT阳性率比较,结直肠癌组高于良性腺瘤组及正常对照组,良性腺瘤组高于正常对照组（P<0.05）;肿瘤低分化、淋巴结转移、脉管/神经侵犯的结直肠癌患者血浆Septin9 DNA甲基化和FOBT阳性率高于肿瘤高分化、中分化及未发生淋巴结转移、未发生脉管/神经侵犯的结直肠癌患者（P<0.05）;随着结直肠癌TNM临床分期升高,血浆Septin9 DNA甲基化及FOBT阳性率升高,差异具有统计学意义（P<0.05）;血浆Septin9 DNA甲基化和FOBT联合诊断结直肠癌的敏感度、曲线下面积（AUC）（87.08%、0.885）均高于两者单独诊断（59.33%、0.744和52.63%、0.643,P<0.05）;血浆Septin9 DNA甲基化诊断结直肠癌的特异度、阳性预测值和阴性预测值分别为89.45%、85.52%和67.68%;FOBT分别为76.38%、70.06%和60.56%,两者联合诊断分别为89.95%、90.10%和86.89%。联合诊断优于单项诊断。结论:随着结直肠病变恶性程度的增加,血浆Septin9 DNA甲基化和FOBT阳性率升高,血浆Septin9 DNA甲基化、FOBT联合检测对结直肠癌具有较高的诊断价值,可作为结直肠癌的实验室诊断指标。