title: Detection of neuroblastoma cells in CD34+ selected peripheral stem cells using a combination of tyrosine hydroxylase nested RT-PCR and anti-ganglioside GD2 immunocytochemistry.
abstract: 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.
authors.affiliation: Department of Haematology Oncology, University Children's Hospital, Tuebingen, Germany.
authors.forename: H N
authors.initials: HN
authors.lastname: Lode
authors.forename: R
authors.initials: R
authors.lastname: Handgretinger
authors.forename: U
authors.initials: U
authors.lastname: Schuermann
authors.forename: G
authors.initials: G
authors.lastname: Seitz
authors.forename: T
authors.initials: T
authors.lastname: Klingebiel
authors.forename: D
authors.initials: D
authors.lastname: Niethammer
authors.forename: J
authors.initials: J
authors.lastname: Beck
mesh_terms: D018952:Antigens, CD34 / Q000032:analysis; D000951:Antigens, Neoplasm / Q000032:analysis; D016465:Bone Marrow Purging / Q000379:methods*; D002469:Cell Separation / Q000379:methods; D018572:Disease-Free Survival; D006412:Hematopoietic Stem Cells / Q000473:pathology*; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D009363:Neoplasm Proteins / Q000032:analysis* / Q000235:genetics; D009447:Neuroblastoma / Q000201:enzymology / Q000276:immunology / Q000473:pathology* / Q000628:therapy; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D014182:Transplantation, Autologous; D014446:Tyrosine 3-Monooxygenase / Q000032:analysis* / Q000235:genetics
chemical_list: D018952:Antigens, CD34; D000951:Antigens, Neoplasm; D009363:Neoplasm Proteins; D014446:Tyrosine 3-Monooxygenase