循环DNA杂合性缺失作为肿瘤标志物在肺癌诊断上的应用
摘要
为了提高肺癌的生存率，寻找特异性好，敏感度高的肿瘤标志物一直是临床研究的重要课题之一。循环DNA是存在于血液（血浆或血清）、滑膜液等体液中的游离DNA，已被广泛应用于基因诊断。杂合性缺失（LOH）是肺癌的频发事件，本课题的研究主要通过测定血浆循环DNA的LOH，以观察循环DNA 的LOH作为一种肿瘤标志物在肺癌诊断上的作用。
我们的工作包括以下两个方面：
一、建立血浆循环DNA的提取方法
分别用酚/氯仿提取法、柱提取法和煮沸法三种方法提取10例血浆标本中的循环DNA，用琼脂糖电泳观察方法能否成功提取循环DNA，并用紫外分光光度法测定其含量和纯度。 结果酚/氯仿提取法、柱提取法和煮沸法分别有10例、10例和4例标本提取的循环DNA可见电泳条带。酚/氯仿提取法提取血浆循环DNA的含量为127±87.9ng/ml，柱提取法为133±88.9ng/ml，煮沸法提取出DNA的含量用紫外分光光度法几乎不可测。三者提取含量经方差分析：差别具有显著性意义（p<0.01），两两比较酚/氯仿提取法、柱提取法之间差别无
显著性（p>0.05），而均与煮沸法有极显著的差别（p<0.01）。酚/氯仿提取法OD260/OD280D比值的均值为1.76 ± 0.12，柱提取法为1.68±0.12，二者经t检验， 差别无显著性意义（p>0.05）。 并以所提取的DNA进行了内参照物β2-微球蛋白的PCR， 观察所提取的DNA是否适合作为PCR反应的模板。酚/氯仿提取法、柱提取法基本能从所有标本中提取出循环DNA，进行PCR的效果较为满意，而煮沸法只有4例标本可进行PCR。三种方法中酚/氯仿提取法所提取的循环DNA的质量和含量都最好，柱提取法次之，煮沸法最差。据此，确定了酚/氯仿提取法作为本研究采用的提取DNA的方法。
二、循环DNA杂合性缺失作为肿瘤标志物在肺癌诊断上的意义
共收集了69例初诊的肺癌患者（后均经病理细胞学确诊）在治疗前的全血标本以及对应组织标本，40例非肺癌肺科住院病人也抽取全血标本，用酚/氯仿提取法提取相应的血浆或组织DNA，并测定了血浆循环DNA的含量。肺癌患者和对照中均含有循环DNA，前者的含量为494±345ng/ml，后者的含量为28±12ng/ml，二者具有十分显著的差异（p<0.01），肺癌患者的血浆循环DNA的含量升高。
选取了D3S1300、D3S1289、D13S171和D17S2179E四个杂合度较高（>95%）的微卫星位点，以PCR-银染法对69例肺癌患者血浆循环DNA和肿瘤组织DNA及对照的血浆循环DNA进行了这四个位点的LOH分析。在69例肺癌患者中，肿瘤DNA的D3S1300位点 LOH检出率为40.6%，D3S1289位点LOH为31.9%，D13S171位点LOH
为33.3%，D17S2179E位点为15.9%，在其相应的血浆中分别有20例检出D3S1300位点 LOH, 检出率为29.0%； 17例检出3S1289位点LOH，检出率为 24.6%；14例检出D13S171位点 LOH, 检出率为20.3%； 8例检出D17S2179E 位点LOH， 检出率为11.6%， 肿瘤组织与血浆DNA的LOH 的阳性率存在显著性差异（p<0.05）。 对照组血浆 DNA各有2例、3例、2例和0例血浆中检出D3S1300位点、D3S1289位点、D13S171位点和D17S2179E位点 LOH，与肺癌组的血浆DNA 的LOH比较具有显著性差别（p< 0.01）。以上结果说明血浆循环DNA存在与原发肿瘤相同的基因改变，可与肿瘤组织DNA一样作为肿瘤标志物。
血浆循环DNA诊断肺癌的效率和所选择的基因有关。血浆循环DNA D3S1300位点、D3S1289位点、D13S171位点和D17S2179E位点的LOH诊断肺癌的特异度和敏感度分别为95%和29.0%，92.5%和24.6%，95%和20.3%，97.5%和11.3%。血浆循环DNA 的D3S1300位点， D13S171位点的LOH与非小细胞性肺癌（NSCLC）的相关性较大；而D3S1289位点LOH与小细胞性肺癌（SCLC）相关性较大；D17S2179E在二者之间差别不大。以>20ng/ml作为阳性判断标准， 癌胚抗原（CEA）诊断肺癌的特异度和敏感度为90.0%和29.0%。与CEA相比，循环DNA单个微卫星位点LOH特异度较高，但敏感度并无优越性，有的甚至低于CEA。
血浆DNA微卫星标志的联合应用可提高对肺癌的诊断效率，以一
个位点出现LOH阳性为诊断标准，微卫星位点联合应用诊断肺癌的效率如下：D3S1300位点+D3S1289位点LOH诊断肺癌的特异度和敏感度分别为87.5%和49.3%； 循环DNA D13S171位点+D3S1289位点LOH为85.0%和37.7%；循环DNA D3S1300位点+D3S1289位点+D17S2179E位点LOH为85.0%和56.2%；循环DNA D13S171位点+D3S1289位点+D17S2179E位点LOH为82.5%和46.4%； 循环DNA D3S1300位点+D13S171位点+D17S2179E位点为82.5%和50.7%； 四个位点联合诊断肺癌的特异度为72.5%，敏感度为65.2%。循环DNA和CEA联合应用诊断肺癌的效率如下：D3S1300位点LOH+CEA的特异度和敏感度分别为87.5%和50.7%，D3S1289位点LOH+CEA为87.5%和42.0%，D3S1300位点LOH+D3S1289位点LOH+CEA为82.5%和66.7%，四个位点的LOH+CEA为77.5%和78.2%。由以上结果可以看出D3S1289位点LOH+D3S1300位点LOH以及D3S1289位点LOH+D3S1300位点LOH+CEA的组合方式较好。
循环DNA LOH对肺癌具有一定的辅助诊断价值，还不能完全满足临床的需要，需要进一步提高检测方法的特异度和灵敏度，以及寻找更特异的标志物。
In order to increase the survival of patients with lung cancer, it is important to find out sensitive and specific tumor markers of lung cancer. Circulating DNA is the free DNA in body fluid (eg: serum or plasma etc.) and it has been used to gene diagnosis. Loss of heterozygosity occurs commonly in human lung cancer. Our study is to investigate the loss of heterozygosity (LOH) of plasma circulating DNA of patients with lung cancer and evaluate the value of circulating DNA as lung cancer tumor marker.
This study included two parts as following:
1. To establish a method to extract plasma DNA.
Three methods, including phenol/chloroform method, QIAamp DNA extraction kit and boiling method, were used to extract plasma DNA of 10 patients. They were sucessfully extracted 10, 10, 4 cases of circulating DNA in 10 plasma samples respectively. DNA purity was identified by the electrophoresis, and DNA concentration was deterimined by the ultraviolet
spectrometer. Plasma DNA concentraction with phenol/ chloroform method was 129±87.9 ng/ml, and it with QIAamp DNA extraction kit was 133±88.5 ng/ml (p>0.05). But there was no detectable DNA concentraction with boiling method by ultraviolet spectrometer. And the ratios of OD260/OD280 of the former two methods were 1.76 ± 0.12 and 1.68±0.12, respectively (p>0.05). It was satisfied to amplify all samples by using the extracted plasma DNA as PCR templet in phenol/chloroform method and QIAamp DNA extraction kit, but it only could amplify 4 cases by using the extracted plasma DNA in boiling method. In conclusion, phenol/chloroform method and QIAamp DNA extraction kit are better than boling method. Therefore, phenol/chloroform method was used to extract circulating DNA in our study.
2. To investigate the loss of heterozygosity (LOH) of plasma circulating DNA in patients with lung cancer and evaluate the diagnostic value of circulating DNA as lung cancer tumor marker
Sixty-nine blood samples and cancer tissues were taken from 69 patients with primary lung cancer before treatment. At the same time, blood samples were also taken in 40 patients with various pulmonary diseases other than lung cancer. Four microsatellite markers D3S1300, D3S1289, D13S171 and D17S2179E were tesed to analysis plasma DNA and cancer tissue DNA LOH in 69 cases of primary lung cancer, and in
plasma DNA of 40 control subjects by PCR and silver staining. LOH ratios of D3S1300 locus, were detected in the tumor tissue and plasma DNA of patients with lung cancer were 40.6% and 29.0%, D3S1289 locus were 31.6 and 24.6%, D13S171 locus were 33.3% and 20.3%, and D17S2179E locus were 15.9% and 11.6 %, respectively. In contrast, there were only 5% D3S1300 LOH, 7.5% D3S1289 LOH, 5% D13S171 LOH and 0.0% D17S2179E LOH in plasma DNA of control group (p < 0.01, vs lung cancer group). In conclusion, there were some gene LOH in circulating DNA of plasma as same as cancer tissue DNA. Plasma circulating DNA LOH could be as a tumor marker to diagnose lung cancer.
The efficiency of plasma DNA to diagnosis lung cancer was correlation with the choosen gene: D3S1300 LOH and D13S171 LOH were correlation with non-small-cell lung cancer (NSCLC), and D3S1289 LOH was correlation with small-cell lung cancer (SCLC), but D17S2179E LOH was correlation with the two ones. The diagnosis specificy and sensitivity of plasma DNA D3S1300 LOH were 95% and 29.0%, D3S1289 LOH were 92.5% and 24.6%, D13S171 LOH were 95% and 20.3%, D17S2179E LOH were 97.5% and 11.3%, and CEA were 90.0% and 29.0%, respectively. Using the combination of microsatellite markers can improve the efficiency of plasma DNA to diagnosis lung cancer: combining the two markers of D3S1300 and D3S1289, the diagnosis
sensitivity improved to 49.3%; and combining the two markers of D13S171 and D3S1289, it improved to 37.7%; combining the three markers of D3S1300 and D3S1289 and D17S2179E, it improved to 56.2%; combining the three markers of D13S171 and D3S1289 and D17S2179E, it improved to 46.4%; and combining the three markers of D3S1300 and
我们的工作包括以下两个方面：
一、建立血浆循环DNA的提取方法
分别用酚/氯仿提取法、柱提取法和煮沸法三种方法提取10例血浆标本中的循环DNA，用琼脂糖电泳观察方法能否成功提取循环DNA，并用紫外分光光度法测定其含量和纯度。 结果酚/氯仿提取法、柱提取法和煮沸法分别有10例、10例和4例标本提取的循环DNA可见电泳条带。酚/氯仿提取法提取血浆循环DNA的含量为127±87.9ng/ml，柱提取法为133±88.9ng/ml，煮沸法提取出DNA的含量用紫外分光光度法几乎不可测。三者提取含量经方差分析：差别具有显著性意义（p<0.01），两两比较酚/氯仿提取法、柱提取法之间差别无
显著性（p>0.05），而均与煮沸法有极显著的差别（p<0.01）。酚/氯仿提取法OD260/OD280D比值的均值为1.76 ± 0.12，柱提取法为1.68±0.12，二者经t检验， 差别无显著性意义（p>0.05）。 并以所提取的DNA进行了内参照物β2-微球蛋白的PCR， 观察所提取的DNA是否适合作为PCR反应的模板。酚/氯仿提取法、柱提取法基本能从所有标本中提取出循环DNA，进行PCR的效果较为满意，而煮沸法只有4例标本可进行PCR。三种方法中酚/氯仿提取法所提取的循环DNA的质量和含量都最好，柱提取法次之，煮沸法最差。据此，确定了酚/氯仿提取法作为本研究采用的提取DNA的方法。
二、循环DNA杂合性缺失作为肿瘤标志物在肺癌诊断上的意义
共收集了69例初诊的肺癌患者（后均经病理细胞学确诊）在治疗前的全血标本以及对应组织标本，40例非肺癌肺科住院病人也抽取全血标本，用酚/氯仿提取法提取相应的血浆或组织DNA，并测定了血浆循环DNA的含量。肺癌患者和对照中均含有循环DNA，前者的含量为494±345ng/ml，后者的含量为28±12ng/ml，二者具有十分显著的差异（p<0.01），肺癌患者的血浆循环DNA的含量升高。
选取了D3S1300、D3S1289、D13S171和D17S2179E四个杂合度较高（>95%）的微卫星位点，以PCR-银染法对69例肺癌患者血浆循环DNA和肿瘤组织DNA及对照的血浆循环DNA进行了这四个位点的LOH分析。在69例肺癌患者中，肿瘤DNA的D3S1300位点 LOH检出率为40.6%，D3S1289位点LOH为31.9%，D13S171位点LOH
为33.3%，D17S2179E位点为15.9%，在其相应的血浆中分别有20例检出D3S1300位点 LOH, 检出率为29.0%； 17例检出3S1289位点LOH，检出率为 24.6%；14例检出D13S171位点 LOH, 检出率为20.3%； 8例检出D17S2179E 位点LOH， 检出率为11.6%， 肿瘤组织与血浆DNA的LOH 的阳性率存在显著性差异（p<0.05）。 对照组血浆 DNA各有2例、3例、2例和0例血浆中检出D3S1300位点、D3S1289位点、D13S171位点和D17S2179E位点 LOH，与肺癌组的血浆DNA 的LOH比较具有显著性差别（p< 0.01）。以上结果说明血浆循环DNA存在与原发肿瘤相同的基因改变，可与肿瘤组织DNA一样作为肿瘤标志物。
血浆循环DNA诊断肺癌的效率和所选择的基因有关。血浆循环DNA D3S1300位点、D3S1289位点、D13S171位点和D17S2179E位点的LOH诊断肺癌的特异度和敏感度分别为95%和29.0%，92.5%和24.6%，95%和20.3%，97.5%和11.3%。血浆循环DNA 的D3S1300位点， D13S171位点的LOH与非小细胞性肺癌（NSCLC）的相关性较大；而D3S1289位点LOH与小细胞性肺癌（SCLC）相关性较大；D17S2179E在二者之间差别不大。以>20ng/ml作为阳性判断标准， 癌胚抗原（CEA）诊断肺癌的特异度和敏感度为90.0%和29.0%。与CEA相比，循环DNA单个微卫星位点LOH特异度较高，但敏感度并无优越性，有的甚至低于CEA。
血浆DNA微卫星标志的联合应用可提高对肺癌的诊断效率，以一
个位点出现LOH阳性为诊断标准，微卫星位点联合应用诊断肺癌的效率如下：D3S1300位点+D3S1289位点LOH诊断肺癌的特异度和敏感度分别为87.5%和49.3%； 循环DNA D13S171位点+D3S1289位点LOH为85.0%和37.7%；循环DNA D3S1300位点+D3S1289位点+D17S2179E位点LOH为85.0%和56.2%；循环DNA D13S171位点+D3S1289位点+D17S2179E位点LOH为82.5%和46.4%； 循环DNA D3S1300位点+D13S171位点+D17S2179E位点为82.5%和50.7%； 四个位点联合诊断肺癌的特异度为72.5%，敏感度为65.2%。循环DNA和CEA联合应用诊断肺癌的效率如下：D3S1300位点LOH+CEA的特异度和敏感度分别为87.5%和50.7%，D3S1289位点LOH+CEA为87.5%和42.0%，D3S1300位点LOH+D3S1289位点LOH+CEA为82.5%和66.7%，四个位点的LOH+CEA为77.5%和78.2%。由以上结果可以看出D3S1289位点LOH+D3S1300位点LOH以及D3S1289位点LOH+D3S1300位点LOH+CEA的组合方式较好。
循环DNA LOH对肺癌具有一定的辅助诊断价值，还不能完全满足临床的需要，需要进一步提高检测方法的特异度和灵敏度，以及寻找更特异的标志物。
In order to increase the survival of patients with lung cancer, it is important to find out sensitive and specific tumor markers of lung cancer. Circulating DNA is the free DNA in body fluid (eg: serum or plasma etc.) and it has been used to gene diagnosis. Loss of heterozygosity occurs commonly in human lung cancer. Our study is to investigate the loss of heterozygosity (LOH) of plasma circulating DNA of patients with lung cancer and evaluate the value of circulating DNA as lung cancer tumor marker.
This study included two parts as following:
1. To establish a method to extract plasma DNA.
Three methods, including phenol/chloroform method, QIAamp DNA extraction kit and boiling method, were used to extract plasma DNA of 10 patients. They were sucessfully extracted 10, 10, 4 cases of circulating DNA in 10 plasma samples respectively. DNA purity was identified by the electrophoresis, and DNA concentration was deterimined by the ultraviolet
spectrometer. Plasma DNA concentraction with phenol/ chloroform method was 129±87.9 ng/ml, and it with QIAamp DNA extraction kit was 133±88.5 ng/ml (p>0.05). But there was no detectable DNA concentraction with boiling method by ultraviolet spectrometer. And the ratios of OD260/OD280 of the former two methods were 1.76 ± 0.12 and 1.68±0.12, respectively (p>0.05). It was satisfied to amplify all samples by using the extracted plasma DNA as PCR templet in phenol/chloroform method and QIAamp DNA extraction kit, but it only could amplify 4 cases by using the extracted plasma DNA in boiling method. In conclusion, phenol/chloroform method and QIAamp DNA extraction kit are better than boling method. Therefore, phenol/chloroform method was used to extract circulating DNA in our study.
2. To investigate the loss of heterozygosity (LOH) of plasma circulating DNA in patients with lung cancer and evaluate the diagnostic value of circulating DNA as lung cancer tumor marker
Sixty-nine blood samples and cancer tissues were taken from 69 patients with primary lung cancer before treatment. At the same time, blood samples were also taken in 40 patients with various pulmonary diseases other than lung cancer. Four microsatellite markers D3S1300, D3S1289, D13S171 and D17S2179E were tesed to analysis plasma DNA and cancer tissue DNA LOH in 69 cases of primary lung cancer, and in
plasma DNA of 40 control subjects by PCR and silver staining. LOH ratios of D3S1300 locus, were detected in the tumor tissue and plasma DNA of patients with lung cancer were 40.6% and 29.0%, D3S1289 locus were 31.6 and 24.6%, D13S171 locus were 33.3% and 20.3%, and D17S2179E locus were 15.9% and 11.6 %, respectively. In contrast, there were only 5% D3S1300 LOH, 7.5% D3S1289 LOH, 5% D13S171 LOH and 0.0% D17S2179E LOH in plasma DNA of control group (p < 0.01, vs lung cancer group). In conclusion, there were some gene LOH in circulating DNA of plasma as same as cancer tissue DNA. Plasma circulating DNA LOH could be as a tumor marker to diagnose lung cancer.
The efficiency of plasma DNA to diagnosis lung cancer was correlation with the choosen gene: D3S1300 LOH and D13S171 LOH were correlation with non-small-cell lung cancer (NSCLC), and D3S1289 LOH was correlation with small-cell lung cancer (SCLC), but D17S2179E LOH was correlation with the two ones. The diagnosis specificy and sensitivity of plasma DNA D3S1300 LOH were 95% and 29.0%, D3S1289 LOH were 92.5% and 24.6%, D13S171 LOH were 95% and 20.3%, D17S2179E LOH were 97.5% and 11.3%, and CEA were 90.0% and 29.0%, respectively. Using the combination of microsatellite markers can improve the efficiency of plasma DNA to diagnosis lung cancer: combining the two markers of D3S1300 and D3S1289, the diagnosis
sensitivity improved to 49.3%; and combining the two markers of D13S171 and D3S1289, it improved to 37.7%; combining the three markers of D3S1300 and D3S1289 and D17S2179E, it improved to 56.2%; combining the three markers of D13S171 and D3S1289 and D17S2179E, it improved to 46.4%; and combining the three markers of D3S1300 and
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16. Sozzi G, Conte D, Mariani L, et al. Analysis of circulating tumour DNA in plasma at diagnosis and during follow-up of lung cancer patients[J]. Cancer Res. 2001, 61:4675-4678
17. Holdenrieder S, Stieber P, Bodenmuller H, et al. Nucleosome in serum of patients withbenign ang malignant disease[J].Int J Cancer. 2001, 95: 114-120
18. Liloglou T, Maloney P, Xinarianos G, et al. Sensitivity and limitations of high throughput fluorscent microsatellite analysis for the detection of alletic imbalance: application in lung tumours [J].Int J oncol.2000(16):5-14
19. 许凯黎，廖美琳，丁安嘉. 非小细胞肺癌分子病理学的研究[J]. 中国肺癌杂志，2001，4（1）：33-36
20. Fournie G J, Lambert P H, Miescher P A, et al. Release of DNA in circulating blood and induction of anti-DNA antibodies after injectionb of bacterial lipolysaccharides [J]. J Exp Med, 1974,140(5): 1189-1206
21. Miyazono F, Takao S, Natsugoe S, et al. Molecular detection of circulating cancer cells during surgery in patients with biliary-pancreatic cancer [J]. Am J Surg, 1999, 177(6): 475-479
22. Hirose T, Kondo K, Takahashi Y, Ishikura H, et al. Frequent microsatellite instability in lung cancer from chromate-exposed workers. Mol Carcinog. 2002, 33(3): 172-180
Gorgoulis VG, Kotsinas A, Zacharatos P, et al. Association of allelic imbalance at locus D13S171 (BRCA2) and p53 alterations with tumor kinetics and chromosomal
23. instability (aneuploidy) in nonsmall cell lung carcinoma. Cancer 2000,89(9): 1933-45
24. Miozzo M, Sozzi G, Musso K, et al. Microsatellite alterations in bronchial and sputum specimens of lung cancer patients. Cancer Res, 1996,56:2285-2288
25. Allan JM, Hardie LJ, Briggs JA, et al. Genetic alterations in bronchial mucosa and plasma DNA from individuals at high risk of lung cancer.Int J Cancer 2001 Feb 1;91(3):359-65
26. Vassilakis DA, Sourvinos G, Markatos M, et al. Microsatellite DNA instability and loss of heterozygosity in pulmonary sarcoidosis[J]. Am J Respir Crit Care Med, 1999, 160(5 Pt 1):1729-1733
27. Pavelic K, Krizanac S, Cacev T, Hadzija MP, et al. Aberration of FHIT gene is associated with increased tumor proliferation and decreased apoptosis-clinical evidence in lung and head and neck carcinomas. Mol Med 2001 ,7(7):442-53