纳米免疫磁珠富集和检测非小细胞肺癌患者循环肿瘤细胞的临床意义

Clinical significance of enrichment and detection of circulating tumor cells in NSCLC patients with immunomagnetic beads

目的探讨利用自主研发的肺癌细胞富集检测试剂盒检测非小细胞肺癌(NSCLc)患者循环肿瘤细胞(CTCs)的可行性及临床价值。方法采用肺癌细胞富集检测试剂盒检测4组标本(A组:Ⅰ期NSCLC组,18例;B组:Ⅱ～Ⅳ期NSCLC组,33例;C组:肺部良性病变组,20例;D组:健康志愿者组,20例)外周血中的肿瘤细胞;同时,采用单纯免疫细胞化学(ICC)法及巢式荧光逆转录聚合酶链反应(RT-PCR)方法检测细胞角蛋白19(CK19 mRNA)、肺特异性X蛋白(LUNX mRNA)的表达作对照。结果采用肺癌细胞富集检测试剂盒检测,A、B、C、D组患者的CK阳性细胞检出率分别为33.3%、60.6%、0%和0%。采用巢式RT-PCR方法检测,A、B、C、D组患者CK19 mRNA表达的阳性率分别为38.9%、63.6%、20.0%和0%,A、B、C、D组患者LUNX mRNA表达的阳性率分别为44.4%、69.7%、10.0%和0%。采用单纯ICC法检测,4组患者的CTCs检出率均为0%。肺癌细胞富集检测试剂盒与单纯ICC法检测比较,差异有统计学意义(P<0.05);肺癌细胞富集检测试剂盒与巢式RT-PCR方法检测比较,差异无统计学意义(P>0.05);A组与B、C、D组患者CK阳性细胞的检出率比较,差异均有统计学意义(P<0.05);阳性率与病理类型、细胞分化程度和临床分期有密切关系(P<0.05)。结论开发的肺癌细胞富集检测试剂盒是检测早期NSCLC患者CTCs的敏感方法,有助于发现隐形微转移、重新确定临床分期及指导患者术后的个体化治疗。

Objective To investigate the feasibility and clinical significance of detection of circulating tumor cells （CTCs） in peripheral blood of NSCLC patients by lung cancer cell immunomagnetic enrichment and detection kit. Methods Four groups of patients （Group A： 18 cases with stage I lung cancer; Group B： 33 cases with stage Ⅱ - Ⅳ lung cancer; Group C： 20 cases with benign pulmonary diseases; Group D： 20 healthy volunteers）were enrolled for detection of CTCs using the lung cancer cell enrichment and detection kit developed by ourselves, and compared with the results obtained using simple ICC method and the detection of CK19 mRNA and LUNX mRNA using nested PCR as control. Results By using lung cancer cell enrichment and detection kit, it was revealed that the detection rates of CK positive cells were 33.3%, 60.6%, 0% and 0% in Group A, B, C and D, respectively. By using nested RT- PCR, the rates of positive expression of CK19 mRNA were 38.9%, 63.6%, 20.0% and 0% in Group A, B, C and D, respectively, and those of LUNX mRNA were 44.4%, 69.7%, 10.0% and 0%, respectively, while the detection rate of CTCs was negative in all groups using simple ICC. There was a significant difference between the results obtained by lung cancer cell enrichment and detection kit and simple ICC method（ P 〈 0.05 ）, while no significant difference was found between the results obtained by lung cancer cell enrichment and detection kit and nested RT-PCR （ P 〉 0.05 ）. There was a significant difference in the detection rates of CK positive cells between group A and groups B, C, D（P 〈0.05）. The micrometastasis in peripheral blood was closely related with pathological types, cell differentiation and TNM staging（P 〈 0.05）. Conclusion The lung cancer cell enrichment and detection kit developed by ourselves is a sensitive and reliable method to detect CTCs in patients with NSCLC. It may be helpful in diagnosis of NSCLC micrometastasis and circulating tumor cells in peripheral blood, re-determination of clinical stage and provide important information for cancer-therapy personalization.