无假基因干扰的CK19 RT-PCR方法检测乳腺癌外周血微转移

Detection of the Micrometastases in Peripheral Blood of Patients with Bre ast Cancer by Means of Reverse Transcriptase-Polymererase Chain Reaction for Cytocreatin 19 Free of Pseudogene Interference

目的:以CK19为基因标志检测乳腺癌患者外周血中播散的肿瘤细胞,并对其临床意义进行评价。方法:采用优化的无假基因干扰的RT-PCR方法检测乳腺癌患者外周血有核细胞的CK19mRNA表达。结果:1)方法可靠性验证:检测30例正常人外周血CK19mRNA表达,结果无假阳性;将乳腺癌细胞系MCF-7以1∶107与正常外周血有核细胞混合,检测CK19mRNA表达,结果无假阴性。2)检测乳腺癌41例,外周血肿瘤细胞阳性检出率26.8%(11/41);乳腺癌患者外周血肿瘤细胞阳性率在患者不同年龄、肿瘤大小、临床分期、组织学分级、ER和PR状态的各组间差异无显著性(P>0.05);淋巴结转移阳性病例外周血肿瘤细胞检出率(38.1%)明显高于淋巴结转移阴性组(15.0%),虽然统计学差异不具有显著性(P=0.10),但提示淋巴结转移阳性的患者肿瘤细胞血行播散的危险性有增加的趋势。结论:以CK19为基因标志采用RT-PCR方法检测乳腺癌患者外周血中的肿瘤细胞可能是乳腺癌有意义的预后指标,可以为治疗方案的选择、治疗疗效的评价及外周血干细胞移植联合大剂量化疗适应证筛选提供重要依据。

Objective:To detect the micrometastases in pe ripheral blood of patients with breast cancer using cytokeratin19(CK19)as a gene marker and discuss the clinical value.Meth ods:Analyzing CK19mRNA ex pression of circulating peripheral blood nucleated cells by the re verse tran sc riptase-polymererase chain reaction method that is free of pseudogene interfer-ence.Re sults:To assess the reliability of the method,30normal peripheral blood samples were detected and it turned out that no CK19mRNA expression wa s false-positive;breast cancer cells MCF-7were mixed with normal peripheral blood nucleated cells at concentrations of1:10 7 ,and no CK19mRNA expression was detected to be false-negative.Of the4 1patients with breast cancer,11(26.8%)were positive for CK19mRNA ex pression of circulating peripheral blood nucle-ated cells,and there was no sig nificant difference relating to ages,tumor sizes,clinical stages, pathological grades,ER and PR(P>0.05).The detectable rate of tu mor cells in peripheral blood of patients with lymph node metastasis(38.1%)i s obviously higher than that of those without lymph node metastasis(15.0% ).Al though the difference is not of statistic sig nif icance(P=0.10)ye t,it does indicate a ten dency to greater danger of dissemination tumor cells o f patients with lymph node metastasis.Conclusions:Detecting mi crometastases i n peripheral blood of patients with breast cancer using cytocreatin19(CK19 )as a gene marker can be used as a prognostic in-dicator,and it may also cont ribute significantly to the decision on therapies,evaluation of treat-ment ef fect,and the selection of the cases suitable for autologous peripheral blood st em cell(PB-SC)transplantation.