流式细胞术检测骨髓微量神经母细胞瘤细胞在临床中的应用

【摘要】目的 阐明神经母细胞瘤（NB）患儿骨髓微量肿瘤病灶(minimal residual disease MRD)的检测方法及其结果对预后的影响。方法 应用CD45-FITC/CD81-PE/CD56-PECy5单抗组合通过流式细胞术(FCM)检测了人NB细胞株TGW的免疫表型并确定本实验室FCM的敏感度；比较BM细胞形态学检查与FCM检测骨髓中的CD45-/ CD81+/ CD56+细胞，分析病程中MRD检测与预后的关系。结果 ⑴ TGW细胞株表达CD56和CD81，不表达CD45，本实验室FCM敏感度为1/104。⑵61例患者共144份标本中细胞形态学提示BM转移者23份;FCM显示表达CD45-/ CD81+/ CD56+细胞的标本数62份，BM细胞形态学检测方法和FCM阳性检出率的比较有显著性差异，（P﹤0.01 X2检验）。⑶比较平均化疗4个疗程后MRD结果与预后关系：31例患儿初诊时BM MRD检测阳性，化疗4个疗程后11例患儿MRD转阴，随访至今无复发及进展，DFS中位时间23mos；另外20例化疗4个疗程后仍为阳性，其中11例复发或进展，包括1例死亡，两组比较有显著性差异（P﹤0.01）。⑷化疗后移植患儿PBSC采集前骨髓MRD结果与预后的相关性：共有19例患儿接受化疗后PBSCT，13例患儿采集时骨髓MRD为阴性，其中2例复发，自移植后始DFS中位时间9mos；6例患儿采集时骨髓MRD为阳性，5例复发，两组比较有显著性差异，（P﹤0.05）。结论 运用FCM检测NB患儿骨髓中的微量肿瘤细胞敏感度高、特异性强，可协助初诊患儿的诊断、评估临床疗效，骨髓中的微量肿瘤细胞残留与神经母细胞瘤预后相关.

The detection of minimal residual disease with flow cytometry and the relationship between prognosis and minimal residual disease in bone marrow in advanced neuroblastoma

【Abstract】 objective This study aimed to detect the method and investigate whether flow cytometry detection of minimal residual disease(MRD) in bone marrow(BM) could predict prognosis and clinically guide the risk-adjusted therapy for neuroblastoma(NB) patients. Methods Human TGW NB cell line were used to detect the immunophenotype of NB cells by flow cytometry (FCM). The FCM sensitivity of the cocktail of CD45-FITC/CD81-PE/CD56-PECy5+ in our lab was tested. The result of MRD with FCM were used to evaluate neuroblastoma contamination in BM at diagnosis, during chemotherapy. Results ⑴ The TGW cell line expressed CD56 and CD81 antigen and did not express CD45. The FCM sensitivity in our lab is 1/104.⑵ In 144 samples from 61 patients, NB cell was found in 23 of them by morphology(BM smear). All of those 23 samples were CD45-/ CD81+/ CD56+ positive by FCM. Thirty-nine BM samples was cytological negative by BM smear but with positive CD45-/ CD81+/ CD56+ by FCM. There was a statistical significant difference between the two methods（P﹤0.01）.⑶ BM samples from 31 patients were positive for neuroblastoma cell by FCM at diagnosis, eleven of them became negative after average 4 courses of chemotherapy. All of those 11 patients remained alive without evidence of disease (median23mos，range8-38mos). In contrast, twenty patients whose BM samples remained positive, eleven of them have relapsed and 1 patient died from disease(median17.5mos，range6-48mos). There was a statistical significant difference in disease-free survival between the two groups（P﹤0.01）. ⑷MRD in BM was tested by FCM before PBSC harvest for 19 advanced neuroblastoma patients. Thirteen was negative, two of them have relapsed. Eleven patients remained alive without evidence of disease (median9mos follow from transplant，range4-30mos). In contrast, another 6 patients whose BM remained positive before PBSC harvest, five of them have relapsed(median10.5mos follow from transplant，range1-21mos). There was a statistically significant difference in disease-free survival after transplant between the two groups（P﹤0.05）.Conclusion The detection of micrometastasis of NB to BM or peripheral blood using FCM had the characteristic of high sensitivity, specificity. It could evaluate the respond to treatment by monitoring the MRD of BM. Positive NB MRD in BM during therapy is an unfavourable factor.