Lode H N, Handgretinger R, Schuermann U, Seitz G, Klingebiel T, Niethammer D, Beck J
Department of Haematology Oncology, University Children's Hospital, Tuebingen, Germany.
Eur J Cancer. 1997 Oct;33(12):2024-30. doi: 10.1016/s0959-8049(97)00243-8.
A sensitive assay was developed for the detection of neuroblastoma cell contamination in CD34+ selected and unseparated peripheral blood stem cells (PBSC) used for autologous transplantation in stage 4 neuroblastoma patients. Specifically, we established a non-radioactive nested cDNA-PCR (nPCR) for detection of tyrosine hydroxylase (TH) gene expression combined with anti-disialoganglioside GD2 immunocytochemistry with the murine monoclonal antibody (MAb) 14G2a. Sensitivities of TH nPCR determined with a number of neuroblastoma cell lines and PBSCs correlated to cell line dependent basal TH gene expression levels and ranged from 1:10(4) to 1:10(6). The sensitivity obtained by immunocytochemistry was 1:10(5). We observed the highest PBSC contamination rate of 47% (18/38) among 38 PBSC specimens exclusively obtained from stage 4 neuroblastoma patients by using TH nPCR and GD2 immunocytochemistry in combination. Furthermore, a clinically applied purging method, CD34+ selection by immunoabsorption (CD34+ purity 42.4%), was used on 16 PBSCs. 10/16 (63%) preparations were contaminated prior to CD34+ selection and 56% (9/16) remained contaminated. A significant reduction of neuroblastoma cell contamination by CD34+ selection was not detectable, but the absolute amount of re-infused tumour cells was decreased due to 100-fold smaller cell counts of CD34+ selected grafts used for transplantation. 22 PBSC preparations were used for transplantation. A Kaplan-Meier analysis showed an event-free survival probability of 0.56 +/- 0.22 (n = 9) in the group with contaminated PBSCs versus 0.88 +/- 0.12 (n = 8) with no detectable neuroblastoma-cell contamination. Our data suggest that the combined use of TH nPCR and GD2 immunocytochemistry is optimal to detect contamination and monitor purging strategies.
我们开发了一种灵敏的检测方法，用于检测4期神经母细胞瘤患者自体移植所用的经CD34+选择和未经分离的外周血干细胞（PBSC）中的神经母细胞瘤细胞污染情况。具体而言，我们建立了一种非放射性巢式cDNA-PCR（nPCR），用于检测酪氨酸羟化酶（TH）基因表达，并结合使用鼠单克隆抗体（MAb）14G2a进行抗双唾液酸神经节苷脂GD2免疫细胞化学检测。用多种神经母细胞瘤细胞系和PBSC测定的TH nPCR灵敏度与细胞系依赖的基础TH基因表达水平相关，范围为1:10(4)至1:10(6)。免疫细胞化学获得的灵敏度为1:10(5)。通过联合使用TH nPCR和GD2免疫细胞化学，我们在仅从4期神经母细胞瘤患者获得的38个PBSC样本中观察到最高的PBSC污染率为47%（18/38）。此外，对16个PBSC采用了一种临床应用的清除方法，即通过免疫吸附进行CD34+选择（CD34+纯度为42.4%）。10/16（63%）的制剂在CD34+选择前被污染，56%（9/16）仍被污染。未检测到CD34+选择能显著降低神经母细胞瘤细胞污染，但由于用于移植的CD34+选择移植物的细胞计数小100倍，回输的肿瘤细胞绝对数量减少。22个PBSC制剂用于移植。Kaplan-Meier分析显示，PBSC受污染组的无事件生存概率为(0.56 \pm 0.22)（(n = 9)），而未检测到神经母细胞瘤细胞污染组为(0.88 \pm 0.1)2（(n = 8)）。我们的数据表明，联合使用TH nPCR和GD2免疫细胞化学是检测污染和监测清除策略的最佳方法。
