悬浮阵列技术检测循环肿瘤细胞基因预测乳腺癌术后复发

Application of suspension array assay to detect marker genes expression of circulating tumor cells for early prediction of breast cancer metastasis

目的建立可以同时检测乳腺癌患者外周血中循环肿瘤细胞多个基因表达水平的悬浮阵列方法,探讨多基因联合检测在乳腺癌术后复发早期预测中的应用价值。方法以悬浮阵列技术检测73例乳腺癌患者外周血循环肿瘤细胞癌基因,并用 COX 比例风险模型寻找影响乳腺癌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表达水平的悬浮阵列方法,基因阳性率从大到小排列的结果为乳腺珠蛋白(hMAM)、人类表皮生长因子受体2(HER2)、细胞角蛋白19(CK19)、乳腺小黏蛋白(SBEM)、肿瘤相关钙信号转导蛋白1(EPG2)、端粒酶逆转录酶(hTERT)、人绒毛膜促性腺激素β亚基(β-HCG)和 B305D 基因,分别为57.5%、57.5%、53.4%、52.1%、31.5%、26.1%、21.9%和15.1%。1个及1个以上基因表达的阳性率为79%,高于血清 CA15-3检测(44%,P<0.05)。SBEM 的表达与临床分期有关(P<0.05);hMAM、SBEM、HER2、雌激素受体(ER)为乳腺癌复发转移的独立预后因素(均 P<0.05)。结论建立的悬浮阵列方法在乳腺癌的预后判断方面具有实用价值。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。

Objective To develop a suspension array assay to detect the expression of multiple genes in the circulating breast cancer cells simultaneously so as to identify the marker genes for human breast cancer metastasis. Methods Peripheral blood samples were obtained from 73 breast cancer patients, including 31 breast cancer metastasis patients, 30 patients with benign breast diseases, and 40 healthy women, and peripheral blood mononuclear cells （PBMCs） were isolated. Total RNA was extracted and cDNA was synthesized. PCR was used to amplify 8 breast cancer-related genes： hMAM, HER2, CK19, SBEM, EPG2, hTERT, β-HGG, and B305D. Suspension array of the PCR products was developed and underwent LuminexTM 100 laser Flow-type analysis to read the fluorescence signal. COX proportional hazard model was used to find the independent prognostic predictors of breast cancer metastasis. Results hMAM expression was detected in 57.5%, HER2 in 57.5%, CK19 in 53.4%, SBEM in 52. 1%, EPG2 in 31.5%, hTERT in 26.1%, β-HCG in 21.9% , and B305D in 15.1% of the blood samples respectively. Compared with serum CA15-3 detection, the multigene detection has higher sensitivity （ P 〈 0.05 ）. The expression of SBEM-mRNA in the peripheral blood was correlated with the stage of breast cancer （P 〈 0. 05 ） ; and hMAM, SBEM, HER2, and ER could be considered as the independent prognostic predictors of breast cancer metastasis （ all P 〈 0. 05 ）. Conclusion The suspension array assay thus developed is practical in diagnosis of the prognosis of breast cancer. The expression of hMAM, SBEM, and HER2 in peripheral blood can be considered as the independent prognostic predictors of breast cancer metastasis.