悬浮阵列技术检测循环肿瘤细胞预测乳腺癌早期复发转移
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摘要
研究背景
乳腺癌是严重威胁妇女健康的疾病,乳腺癌的早期检出能够降低乳腺癌病人的死亡率。乳腺癌病人死亡与早期肿瘤复发转移密切相关,而早期转移往往发生在乳腺癌的较早阶段。而循环肿瘤细胞具有原发肿瘤的特征,肿瘤转移前后都可以检测到循环肿瘤细胞。乳腺癌循环肿瘤细胞的检测意义重大,包括乳腺癌的早期诊断,复发转移的早期诊断,指导术后治疗等多个方面。因此通过改善血液中隐蔽转移肿瘤细胞的检测方法以做出乳腺癌早期复发转移的诊断,对于病人的预后以及治疗措施的选择具有重大意义。
悬浮阵列是一种多功能的生物芯片平台,它有机地整合了编码微珠、激光技术、应用流体学、高速数字信号处理器和计算机运算法则,造就了其检测的高特异性和高灵敏度,可广泛应用于DNA杂交、免疫分析、基因及蛋白质表达谱分子检测等多个研究领域,也是目前唯一被美国FDA批准用于临床诊断的生物芯片平台。
本课题基于Luminex?100激光流式分析仪平台,建立可以同时检测乳腺癌患者外周血中8种循环肿瘤细胞基因(包括hMAM、HER2、CK-19、SBEM、EPG2、hTERT、β-HCG和B305D)表达水平的悬浮阵列方法,以探讨多基因联合检测在乳腺癌早期复发转移诊断中的应用价值。
方法
1.参考国外文献我们选择了8种循环乳腺癌细胞相关基因进行研究,并使用Primer 5.0软件设计引物和探针序列;
2.将人乳腺癌细胞株MCF7加至健康人外周血中,制成含有不同浓度乳腺癌细胞的外周血标本模型,用于建立可以同时检测8种循环肿瘤细胞基因表达水平的逆转录PCR方法;
3.合成好的探针用于包被Luminex微珠,制备Luminex杂交液;
4.建立可以同时检测8种循环肿瘤细胞基因表达水平的悬浮阵列方法,并进行方法学评价;
5.收集乳腺癌患者外周血标本,以悬浮阵列技术检测乳腺癌患者外周血循环肿瘤细胞癌基因;
6.分析乳腺癌相关基因表达与肿瘤组临床病理因素、临床分期的关系,并用Cox比例风险模型寻找乳腺癌术后复发转移的独立预后因素。
结果
成功建立了可以同时检测乳腺癌患者外周血中8种循环肿瘤细胞基因(包括HER2、CK-19、hMAM、SBEM、hTERT、β-HCG、EGP2和B305D-C)表达水平的悬浮阵列测定方法,本方法检测限为从1ml全血中可以检测到50个MCF7细胞。悬浮阵列方法特异性评估分析结果显示,每种微珠上包被的特异性探针仅和相应基因的PCR产物杂交,没有发现交叉反应,未检测到假阴性和假阳性的结果。在全部73例乳腺癌患者中,单个基因检测的阳性率从大到小排列的结果为hMAM、HER2、CK19、SBEM、EPG2、hTERT、β-HCG和B305D,分别为58%、58%、53%、52%、31%、26%、22%和15%;多基因联合检测与血清CA15-3比较,检测乳腺癌预后有更高的灵敏度(P<0.05);8个乳腺癌相关基因的表达与患者的年龄、肿瘤大小和肿瘤类型无关(P>0.05),其中只有SBEM的表达与临床分期有比较明显的相关性(X2=8.310,P=0.040<0.05)。Cox比例风险模型发现hMAM、SBEM、HER2、ER为乳腺癌复发转移的独立预后因素,其相对危险度(HR)分别为2.66、2.88、2.63和0.43。
结论
建立的悬浮阵列方法在乳腺癌的预后判断和临床治疗方面具有很大的实用价值。外周血循环肿瘤细胞hMAM、SBEM和HER2的表达可以作为分子标记物预测乳腺癌的复发转移。
Backgroud and Objective
Early detection of breast cancer can reduce the morbidity and mortality of breast cancer patients . Breast cancer mortality occurs in a substantial portion of breast cancer patients and is increasingly linked to early metastases, which are often occult at the time of primary diagnosis. Because undetected micrometastases can contribute to the failure of primary treatment, their identification in patients with early breast cancer relapse may have a substantial impact on prognosis and treatment choice for these patients. It is very convenient for collecting peripheral blood samples to detect occult circulating peripheral tumor cells, which is more favourable for clinical monitoring consecutively. Thus, improved direct identification of these occult metastases in blood offers a critical opportunity to optimize management of breast cancer patients.
Microsphere-based suspension array technologies, such as the Luminex xMAP system, offer a new platform for high-throughput nucleic acid detection which is being used in a variety of applications. Some benefits of suspension array technology include rapid data acquisition, excellent sensitivity and specificity, and multiplexed analysis capability. As compared to planar microarrays, suspension arrays have the benefits of ease of use, low cost, statistical superiority, faster hybridization kinetics and more flexibility in array preparation.
So we want to develop a Suspension array assay for the detection of marker genes expression for human circulating breast cancer cells ,in order to analyze the independent prognostic predictors of breast cancer metastasis.
Methods
1. The Primer 5.0 software is used to design the primers and probes sequences for marker genes for human circulating breast cancer cells.
2. In order to prepare Luminex hybridization liquid, the specific probes was used to coat Luminex beads.
3. The human MCF7 cells is used as a model to develop a Suspension array assay for the detection of marker genes expression for human circulating breast cancer cells.
4. Then our suspension array is used to examine the multigene expression in PBMC of 73 breast cancer patients,including the breast cancer relapse patients.
5. COX proportional hazard modle was used to find the independent prognostic predictors of breast cancer relapse.
Results
We develop successfully a suspension array assay for the detection of marker genes expression for human circulating breast cancer cells (including hMAM、HER2、CK19、SBEM、EPG2、hTERT、β-HCG and B305D) ,and the detection limit of this assay is to detect 50 MCF7 tumor cells per milliliter peripheral blood. Resolution of the mix of amplified products was achieved by PCR product hybridization to corresponding probe sequences, attached to unique sets of fluorescent beads. The assay was optimized to allow for maximum sensitivity in a multiplexed format. And there were no false-positive results or false-negative results being detected. Gene expression analysis of cells collected from blood of 73 women with breast cancer was performed with the multigene RT-PCR and suspension array assay. hMAM expression was detected in 58%, HER2 in 58%, CK19 in 53%, SBEM in 52%, EPG2 in 31%, hTERT in 26%,β-HCG in 22% and B305D in 15% of the blood samples, respectively. But detection in peripheral blood of any of the eight marker genes included in the multigene assay is not significantly associated with increasing age, tumor size and tumor type.And we discover that hMAM, SBEM, HER2 and ER could be considered as the independent prognostic predictors of breast cancer relapse by using Cox PH model, and the Relative hazard(HR) of hMAM, SBEM, HER2 and ER is 2.66,2.88,2.63 and 0.43, respectively.
Conclusion
The sensitivity and specificity of the suspension array assay for the detection of marker genes expression for human circulating breast cancer cells are rather high, and the characterization of circulating tumor cells based on the detection of breast-specific marker genes with a suspension array assay has good potential for early detection of early detection of breast cancer relapse. And the expression of hMAM、SBEM and HER2 in peripheral blood can be considered as the independent prognostic predictors of breast cancer metastasis.
乳腺癌是严重威胁妇女健康的疾病,乳腺癌的早期检出能够降低乳腺癌病人的死亡率。乳腺癌病人死亡与早期肿瘤复发转移密切相关,而早期转移往往发生在乳腺癌的较早阶段。而循环肿瘤细胞具有原发肿瘤的特征,肿瘤转移前后都可以检测到循环肿瘤细胞。乳腺癌循环肿瘤细胞的检测意义重大,包括乳腺癌的早期诊断,复发转移的早期诊断,指导术后治疗等多个方面。因此通过改善血液中隐蔽转移肿瘤细胞的检测方法以做出乳腺癌早期复发转移的诊断,对于病人的预后以及治疗措施的选择具有重大意义。
悬浮阵列是一种多功能的生物芯片平台,它有机地整合了编码微珠、激光技术、应用流体学、高速数字信号处理器和计算机运算法则,造就了其检测的高特异性和高灵敏度,可广泛应用于DNA杂交、免疫分析、基因及蛋白质表达谱分子检测等多个研究领域,也是目前唯一被美国FDA批准用于临床诊断的生物芯片平台。
本课题基于Luminex?100激光流式分析仪平台,建立可以同时检测乳腺癌患者外周血中8种循环肿瘤细胞基因(包括hMAM、HER2、CK-19、SBEM、EPG2、hTERT、β-HCG和B305D)表达水平的悬浮阵列方法,以探讨多基因联合检测在乳腺癌早期复发转移诊断中的应用价值。
方法
1.参考国外文献我们选择了8种循环乳腺癌细胞相关基因进行研究,并使用Primer 5.0软件设计引物和探针序列;
2.将人乳腺癌细胞株MCF7加至健康人外周血中,制成含有不同浓度乳腺癌细胞的外周血标本模型,用于建立可以同时检测8种循环肿瘤细胞基因表达水平的逆转录PCR方法;
3.合成好的探针用于包被Luminex微珠,制备Luminex杂交液;
4.建立可以同时检测8种循环肿瘤细胞基因表达水平的悬浮阵列方法,并进行方法学评价;
5.收集乳腺癌患者外周血标本,以悬浮阵列技术检测乳腺癌患者外周血循环肿瘤细胞癌基因;
6.分析乳腺癌相关基因表达与肿瘤组临床病理因素、临床分期的关系,并用Cox比例风险模型寻找乳腺癌术后复发转移的独立预后因素。
结果
成功建立了可以同时检测乳腺癌患者外周血中8种循环肿瘤细胞基因(包括HER2、CK-19、hMAM、SBEM、hTERT、β-HCG、EGP2和B305D-C)表达水平的悬浮阵列测定方法,本方法检测限为从1ml全血中可以检测到50个MCF7细胞。悬浮阵列方法特异性评估分析结果显示,每种微珠上包被的特异性探针仅和相应基因的PCR产物杂交,没有发现交叉反应,未检测到假阴性和假阳性的结果。在全部73例乳腺癌患者中,单个基因检测的阳性率从大到小排列的结果为hMAM、HER2、CK19、SBEM、EPG2、hTERT、β-HCG和B305D,分别为58%、58%、53%、52%、31%、26%、22%和15%;多基因联合检测与血清CA15-3比较,检测乳腺癌预后有更高的灵敏度(P<0.05);8个乳腺癌相关基因的表达与患者的年龄、肿瘤大小和肿瘤类型无关(P>0.05),其中只有SBEM的表达与临床分期有比较明显的相关性(X2=8.310,P=0.040<0.05)。Cox比例风险模型发现hMAM、SBEM、HER2、ER为乳腺癌复发转移的独立预后因素,其相对危险度(HR)分别为2.66、2.88、2.63和0.43。
结论
建立的悬浮阵列方法在乳腺癌的预后判断和临床治疗方面具有很大的实用价值。外周血循环肿瘤细胞hMAM、SBEM和HER2的表达可以作为分子标记物预测乳腺癌的复发转移。
Backgroud and Objective
Early detection of breast cancer can reduce the morbidity and mortality of breast cancer patients . Breast cancer mortality occurs in a substantial portion of breast cancer patients and is increasingly linked to early metastases, which are often occult at the time of primary diagnosis. Because undetected micrometastases can contribute to the failure of primary treatment, their identification in patients with early breast cancer relapse may have a substantial impact on prognosis and treatment choice for these patients. It is very convenient for collecting peripheral blood samples to detect occult circulating peripheral tumor cells, which is more favourable for clinical monitoring consecutively. Thus, improved direct identification of these occult metastases in blood offers a critical opportunity to optimize management of breast cancer patients.
Microsphere-based suspension array technologies, such as the Luminex xMAP system, offer a new platform for high-throughput nucleic acid detection which is being used in a variety of applications. Some benefits of suspension array technology include rapid data acquisition, excellent sensitivity and specificity, and multiplexed analysis capability. As compared to planar microarrays, suspension arrays have the benefits of ease of use, low cost, statistical superiority, faster hybridization kinetics and more flexibility in array preparation.
So we want to develop a Suspension array assay for the detection of marker genes expression for human circulating breast cancer cells ,in order to analyze the independent prognostic predictors of breast cancer metastasis.
Methods
1. The Primer 5.0 software is used to design the primers and probes sequences for marker genes for human circulating breast cancer cells.
2. In order to prepare Luminex hybridization liquid, the specific probes was used to coat Luminex beads.
3. The human MCF7 cells is used as a model to develop a Suspension array assay for the detection of marker genes expression for human circulating breast cancer cells.
4. Then our suspension array is used to examine the multigene expression in PBMC of 73 breast cancer patients,including the breast cancer relapse patients.
5. COX proportional hazard modle was used to find the independent prognostic predictors of breast cancer relapse.
Results
We develop successfully a suspension array assay for the detection of marker genes expression for human circulating breast cancer cells (including hMAM、HER2、CK19、SBEM、EPG2、hTERT、β-HCG and B305D) ,and the detection limit of this assay is to detect 50 MCF7 tumor cells per milliliter peripheral blood. Resolution of the mix of amplified products was achieved by PCR product hybridization to corresponding probe sequences, attached to unique sets of fluorescent beads. The assay was optimized to allow for maximum sensitivity in a multiplexed format. And there were no false-positive results or false-negative results being detected. Gene expression analysis of cells collected from blood of 73 women with breast cancer was performed with the multigene RT-PCR and suspension array assay. hMAM expression was detected in 58%, HER2 in 58%, CK19 in 53%, SBEM in 52%, EPG2 in 31%, hTERT in 26%,β-HCG in 22% and B305D in 15% of the blood samples, respectively. But detection in peripheral blood of any of the eight marker genes included in the multigene assay is not significantly associated with increasing age, tumor size and tumor type.And we discover that hMAM, SBEM, HER2 and ER could be considered as the independent prognostic predictors of breast cancer relapse by using Cox PH model, and the Relative hazard(HR) of hMAM, SBEM, HER2 and ER is 2.66,2.88,2.63 and 0.43, respectively.
Conclusion
The sensitivity and specificity of the suspension array assay for the detection of marker genes expression for human circulating breast cancer cells are rather high, and the characterization of circulating tumor cells based on the detection of breast-specific marker genes with a suspension array assay has good potential for early detection of early detection of breast cancer relapse. And the expression of hMAM、SBEM and HER2 in peripheral blood can be considered as the independent prognostic predictors of breast cancer metastasis.
引文
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