神经母细胞瘤循环肿瘤细胞分离与鉴定方法的建立及临床应用

Establishment and clinical application for isolation and identification of circulating tumor cells in neuroblastoma

目的建立一种神经母细胞瘤(NB)循环肿瘤细胞(CTCs)的分离和鉴定方法,初步探讨其测定NB患者CTCs的临床价值。方法应用CD56免疫磁珠阳性分选结合免疫荧光染色方法,对IMR32细胞和20例非血液肿瘤疾病患儿的外周血样本进行处理,通过统计捕获到的DAPI^+/GD2^+/CD45^-细胞数目,计算该方法分离和鉴定NB细胞的敏感性和特异性;检测12例初发晚期NB患儿治疗前及2疗程化疗后外周血中的CTCs数量。结果 10个以内IMR32细胞组,CTCs捕获的敏感性为(80±6.73)%;10～100个IMR32细胞组,CTCs捕获的敏感性为(79.14±3.97)%。20例非血液肿瘤疾病患儿的外周血中均未检测到DAPI^+/GD2^+/CD45^-细胞的存在,提示该方法捕获CTCs的特异性为100%。12例Ⅲ期或Ⅳ期NB患儿,治疗前有9例检测出CTCs,阳性率为75%;化疗2个疗程后,9例患儿CTCs数目均有不同程度减少,该动态变化趋势与患儿血清NSE水平、24h尿VMA水平及骨髓MRD变化相吻合,而影像学评估显示3例为部分反应(PR),6例为混合反应(MR),3例无反应(NR)。结论免疫磁珠阳性分选结合免疫荧光染色的方法对于NB患儿CTCs的捕获具有良好的敏感性和特异性,CTCs数目动态变化比影像学检测可更灵敏地反映疗效,该方法为进一步探讨CTCs在NB儿童疗效评估及复发风险预测中的价值提供了技术手段。

Objective To establish and optimize a sensitive and specific method for isolation and identification of circulating tumor cells （CTCs） in peripheral blood of children with neuroblastoma, and to evaluate its sensitivity, specificity and clinical value. Methods Blood samples spiked with neuroblastoma IMR32 cells （range from 1-i0 and i0-i00 ） and another 20 blood samples from non- hematology/oncology patients were sorted with CD56 magnetic beads positively and stained with antibodies labeled with fluorescence. The sensitivity and specificity of this method was calculated by counting the number of DAPI ＋/GD2 ＋/CD45- cells with fluorescent microscope. The numbers of CTCs from 12 children with neuroblastoma were explored at diagnosis and after 2 courses of chemotherapy. Results In 1-10 IMR32 cells group, the sensitivity of this method was （80±6.73）% ;while in 10-100 IMR32 cells group, the sensitivity was （79.14 ± 3.97 ） %. No DAPI ＋/GD2 ＋/CD45 - cells were detected in the blood samples from 20 non-tumor patients, suggesting that its specificity was 100%. Among 12 patients with Stage III or IV neuroblastoma, CTCs were separated successfully from 9 cases before treatment, so the positive rate of CTCs was 75%. After 2 courses of chemotherapy, the numbers of CTCs decreased obviously. The tendency of CTCs was in accordance with the levels of serum NSE, 24h urine VMA and minimal residual disease of bone marrow. The imaging assessment showed that 3 patients had partial response, 6 cases had mixed response and 3 children had no response. Conclusions A combination of immunomagnetic beads positive sorting and immunofluorescence staining techniques has a good sensitivity and specificity in the CTCs capture from patients with neuroblastoma. The changing trend of CTCs number could better reflect the curative effect than imaging. This method may provide a practicable technology to estimate the value of CTCs on the response assessment and recurrence prediction in neuroblastoma.