摘要
消化道肿瘤患者血浆DNA定量检测及临床特征分析
背景
近年来消化道肿瘤在世界各国的发病率和病死率均迅速上升,中国是消化道肿瘤的高发国家之一,根据中国肿瘤登记处恶性肿瘤发病资料统计分析,胃癌、食管癌在威胁我国居民健康的主要恶性肿瘤中分列第2、3位,仅次于肺癌。目前,对于消化道肿瘤的诊断较为敏感的检测方法为内镜检查,然而由于其成本高,检查具侵袭性,患者不易耐受,不适于广大人群的筛查。因此,创伤性小,敏感性高的检测方法对于消化道肿瘤的早期筛查显得尤为重要。
循环DNA(circulating DNA),又称为游离DNA(cell-free DNA),是存在于血液(血浆或血清)、脑脊液以及滑膜液等体液中的细胞外DNA。早在1948年,在Watson和Crick证明DNA的双螺旋结构之前,Mandel等就在病人和健康人群的血浆中检测到了循环DNA,血浆中循环DNA,简称血浆DNA。但这一成果直到1977年Leon报道了肿瘤病人血清循环DNA浓度明显高于非肿瘤病人后才受到广泛的关注。此后,有关外周血循环DNA在肿瘤的诊断、治疗及预后评估方面的报道逐渐增多。研究证实,健康人体的血清及血浆中只含有极少量的循环DNA,而肿瘤病人的外周血循环DNA主要来源于肿瘤细胞的坏死或凋亡、微转移灶或循环肿瘤细胞的裂解和增值旺盛的肿瘤细胞所释放。在肿瘤病人外周血循环DNA中可检测到与原发肿瘤细胞相一致的分子生物学特性。早期研究显示血浆DNA有可能成为一种新的肿瘤早期诊断的标志物。近年来,日益增多的研究表明,肿瘤在生长过程中能持续释放肿瘤细胞DNA进入外周血液,肿瘤患者外周血中循环DNA的浓度增加,伴有转移的肿瘤患者其浓度更高于早期恶性肿瘤患者。
血浆DNA相关研究已成为热点。然而,目前关于消化道肿瘤患者血浆DNA水平及其与临床特征的分析,尚缺少系统性研究的报道。
目的
本研究对全消化道肿瘤患者血浆DNA含量进行了检测,并分析患者血浆DNA水平与临床病理特征之间的关系,探讨血浆DNA定量检测在消化道肿瘤早期筛查,早期诊断方面的价值。
方法
177名消化道肿瘤患者来自2007年11月至2008年10月南京医科大学第一附属医院,其中食管癌患者100例(男性66名,女性34名,年龄37~86岁,中位数:59岁);贲门癌患者15名(男性9名,女性6名,年龄57~76岁,中位数:63岁);胃癌患者27名(男性21名,女性6名,年龄36~78岁,中位数:59岁);结肠癌患者15名(男性8名,女性7名,年龄40~74岁,中位数:56岁);直肠癌患者20名(男性10名,女性10名,年龄37~86岁,中位数:61岁),采集肘静脉血,EDTA-K2抗凝,于室温1 600×g离心10min,上层血浆再于4℃16 000×g离心10min,分装上层血浆每管200μl于-70℃保存待用。采用含内参照双重荧光定量PCR进行血浆DNA定量检测,肿瘤的分期分级依照AJCC癌症分期手册(第六版)进行。250例健康对照者为本院同期门诊体检健康者,男性125名,女性125名,年龄22~77岁,中位数:42岁。
结果
所有计量资料以中位数(四分位数区间)形式表示。血浆DNA含量在健康对照组为18.2 (13.9~26.2) ng/ml;各种消化道肿瘤患者血浆DNA含量均显著高于健康对照:食管癌患者血浆DNA含量为42.2(27.0~63.6)ng/ml,P<0.001;贲门癌患者血浆DNA含量为31.9(19.9~52.9)ng/ml,P=0.003;胃癌患者血浆DNA含量106.4(73.2~141.6)ng/ml,P<0.001;结肠癌患者血浆DNA含量49.9(26.6~73.2) ng/ml,P<0.001;直肠癌患者血浆DNA含量40.8(27.1~55.5) ng/ml,P<0.001。在食管癌与结肠癌患者中血浆DNA含量与肿瘤临床分期和分级有关,而对于直肠癌患者,血浆DNA含量与肿瘤临床分期和肿瘤大小有关。贲门癌与胃癌患者血浆DNA含量在不同年龄、性别、肿瘤大小、淋巴结侵犯、AJCC分期、分化等级分组间无统计学差异,但晚期、分化差的肿瘤患者血浆DNA水平有升高的趋势。使用非参数ROC分析血浆DNA定量对消化道肿瘤患者的诊断效能,其曲线下面积(AUC)及诊断敏感度均优于传统指标血清CEA。尤其是对于胃癌患者,血浆DNA定量具有最高的诊断效能(敏感度:96.3%,特异度:100%,曲线下面积:0.990)。
结论
与健康对照组相比,恶性肿瘤患者血浆DNA水平明显升高,血浆DNA含量检测对于消化道肿瘤的诊断有一定的价值,是肿瘤诊断的一个重要生物学指标。此外,血浆DNA水平在胃癌早期患者中也显著升高,提示血浆DNA检测对该肿瘤早期诊断具有重要价值。采用ROC曲线来评价血浆DNA对于消化道肿瘤的诊断效能,本研究结果显示血浆DNA对于消化道肿瘤的诊断优于传统肿瘤标志物血清CEA,提示血浆DNA检测对于消化道肿瘤的诊断具有较高的价值。检测血浆DNA对消化道肿瘤的筛查,早期诊断,判断肿瘤侵袭与转移具有重要价值。
Background
Gastrointestinal malignancy is one of the most common causes of cancer-related death worldwide, and China is one of the countries with a high incidence of the disease. Development of efficient diagnostic methods to enable their early detection plays an essential role in increasing the survival rate of patients with these diseases. Although endoscopy is considered the most sensitive screening tool for gastrointestinal malignancies, its use is limited due to its considerable cost and risk, and patients’lack of acceptance of the invasive procedure. In China, a developing country with a huge population, endoscopy is not suitable for large screening programs. Therefore, reliable noninvasive test, preferably blood test, for screening and diagnostic purposes are obviously needed. Plasma DNA, also known as circulating cell-free DNA, is a kind of deoxyribonucleic acid existing in the bloodstream but out of blood cells. In recent years, many studies demonstrated that increased concentrations of cell-free DNA in plasma or serum had been found in patients suffering from different types of cancer. However, the quantification and clinical-pathological characteristics of plasma DNA in gastrointestinal malignancies have not yet been investigated in a systemic research.
Objective
The aim of this study was to quantify the cell-free DNA in plasma samples from patients with different types of gastrointestinal malignancies and to attempt to correlate the resultants with the clinical-pathological parameters.
Methods
A total of 177 patients suffer from gastrointestinal malignancies and 250 healthy controls from the First Affiliated Hospital of Nanjing Medical University were enrolled in the study. DNA was extracted from plasma samples of these patients and healthy controls by using the plasma DNA magnetic bead extraction kit. The quantity of plasma DNA was determined by using duplex real-time quantitative PCR with internal control. Carcinoembryonic antigen (CEA) was determined by radioimmunoassay. The performance of the test was evaluated with a ROC curve. The relationship between the DNA concentration and main demographic, clinical and pathological variables was examined.
Results
The median concentration of the plasma DNA in the healthy controls was 18.2 ng/ml. A significant difference was seen in the median plasma DNA between different types of gastrointestinal malignancies and the healthy controls: esophageal cancer, Median (ME): 42.2 ng/ml, Interquartile range (IQR): 27.0~63.6 ng/ml, P< 0.001; gastric cardiac carcinoma, ME: 31.9 ng/ml, IQR: 19.9~52.9 ng/ml, P=0.003; gastric cancer, ME: 106.4 ng/ml, IQR: 73.2~141.6 ng/ml, P< 0.001; colon cancer, ME: 49.9 ng/ml, IQR: 26.6~73.2 ng/ml, P< 0.001; and rectal cancer, ME: 40.8 ng/ml, IQR: 27.1~55.5 ng/ml, P< 0.001. Plasma DNA was significantly associated with pathological stage and grading in esophageal cancer and colon cancer. And in rectal cancer, the concentration of plasma DNA correlated with pathological stage and tumor size (P<0.05). No association was observed between plasma DNA values and the various clinical-pathological parameters in gastric cardiac carcinoma and gastric cancer. But a trend was found that plasma DNA increased in advanced stage and poor differentiation. The areas under the ROC curves (AUC ROC) assessing plasma DNA concentration were significantly larger than serum CEA for the five types of cancers. Plasma DNA was more sensitive than CEA in detecting gastrointestinal malignancies. In gastric cancer, compared with serum CEA, plasma DNA had the highest diagnostic efficiency (sensitivity 96.3%, specificity 100%, AUC ROC = 0.990).
Conclusion
Plasma DNA values are significantly higher in patients with gastrointestinal malignant neoplasm than the healthy controls, which have practical implications in screening programs and monitoring progression of gastrointestinal malignancies, especially in gastric cancer.
引文
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